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Irlandia 2019 Kuliah 15 - Biologi

Irlandia 2019 Kuliah 15 - Biologi


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Pengantar regulasi gen

Regulasi adalah semua tentang pengambilan keputusan. tisu).

Karena subjek regulasi adalah topik studi biologi yang sangat dalam dan luas, di Bis2a kami tidak mencoba membahas setiap detail - terlalu banyak. Sebaliknya, seperti yang telah kami lakukan untuk semua topik lainnya, kami mencoba untuk fokus pada (a) menguraikan beberapa konstruksi logis inti dan pertanyaan yang harus Anda miliki ketika Anda mendekati skenario APAPUN yang melibatkan regulasi, (b) mempelajari beberapa kosakata umum dan mekanisme di mana-mana dan (c) menelaah beberapa contoh konkrit yang mengilustrasikan poin-poin yang dibuat dalam a dan b.

Ekspresi gen

pengantar

Semua sel mengontrol kapan dan seberapa banyak masing-masing gennya diekspresikan. Pernyataan sederhana ini - yang dapat diturunkan hanya dari mengamati perilaku seluler - memunculkan banyak pertanyaan yang dapat kita mulai susun dengan menggunakan rubrik Tantangan Desain.

Mencoba mendefinisikan "ekspresi gen"

Namun, hal pertama yang perlu kita lakukan adalah mendefinisikan apa artinya ketika kita mengatakan bahwa sebuah gen "diekspresikan". Jika gen mengkodekan protein, seseorang mungkin secara masuk akal mengusulkan bahwa "ekspresi" gen berarti berapa banyak protein fungsional yang dibuat. Tetapi bagaimana jika gen tersebut tidak mengkodekan protein, melainkan beberapa RNA fungsional. Kemudian, dalam hal ini, "ekspresi mungkin berarti berapa banyak RNA fungsional yang dibuat. Namun orang lain mungkin secara wajar menyarankan bahwa "ekspresi" hanya mengacu pada langkah awal dalam membuat salinan informasi genom. Dengan definisi itu, seseorang mungkin ingin menghitung berapa banyak transkrip panjang penuh yang dibuat. Apakah itu jumlah produk akhir yang dikodekan oleh informasi genomik atau apakah jumlah pembacaan informasi yang penting untuk menggambarkan "ekspresi" dengan benar. Sayangnya, dalam praktiknya kami sering menemukan bahwa definisi tergantung pada konteks diskusi. Ingatlah hal itu. Demi memastikan bahwa kita berbicara tentang hal yang sama, di Bis2A kita akan mencoba menggunakan istilah "ekspresi" terutama untuk menggambarkan penciptaan produk fungsional akhir Tergantung pada kasus tertentu, produk akhir dapat berupa spesies protein atau RNA.

Tantangan desain mengatur ekspresi gen

Untuk mendorong diskusi ini dari perspektif tantangan desain, kita dapat secara formal menetapkan bahwa "masalah besar" yang ingin kita abaikan adalah mengatur kelimpahan protein dalam sel. Masalah: Kelimpahan setiap protein fungsional harus diatur. Kita kemudian dapat mulai dengan mengajukan submasalah:

Mari kita luangkan waktu sejenak, pertama untuk memuat ulang beberapa ide. Proses ekspresi gen membutuhkan beberapa langkah tergantung pada bagaimana nasib produk akhir nantinya. Dalam kasus RNA struktural dan regulasi (yaitu tRNA, rRNA, snRNA, dll.) prosesnya mengharuskan gen ditranskripsi dan pemrosesan pasca-transkripsi yang diperlukan berlangsung. Dalam kasus gen penyandi protein, transkrip juga harus diterjemahkan ke dalam protein dan jika diperlukan, modifikasi protein juga harus dilakukan. Tentu saja, baik transkripsi maupun translasi adalah proses multi-langkah dan sebagian besar sub-langkah tersebut juga merupakan situs kontrol yang potensial.

Oleh karena itu, beberapa submasalah mungkin:

  1. Masuk akal untuk mendalilkan bahwa harus ada beberapa mekanisme untuk mengatur langkah pertama dari proses multi-langkah ini, inisiasi transkripsi (baru saja memulai). Jadi, kita dapat menyatakan, "kita memerlukan mekanisme untuk mengatur inisiasi transkripsi." Kita juga dapat mengubah ini menjadi pertanyaan dan bertanya, "bagaimana inisiasi transkripsi dapat dicapai"?
  2. Kita dapat menggunakan pemikiran serupa untuk menyatakan, "kita memerlukan mekanisme untuk mengatur akhir transkripsi" atau untuk bertanya "bagaimana transkripsi dihentikan?"
  3. Dengan menggunakan konvensi ini kita dapat menyatakan, "kita perlu mengatur inisiasi penerjemahan dan penghentian penerjemahan".
  4. Kami hanya berbicara tentang sintesis protein dan RNA. Cukup beralasan juga untuk menyatakan, "kita membutuhkan mekanisme untuk mengatur degradasi RNA dan protein."

Berfokus pada transkripsi

Dalam kursus ini kita mulai dengan berfokus terutama pada pemeriksaan beberapa masalah/pertanyaan pertama, regulasi inisiasi dan terminasi transkripsi - dari informasi genomik ke RNA fungsional, baik siap apa adanya (misalnya dalam kasus RNA fungsional) atau siap pakai. untuk terjemahan. Hal ini memungkinkan kita untuk memeriksa beberapa konsep dasar mengenai regulasi ekspresi gen dan untuk memeriksa beberapa contoh nyata dari konsep-konsep tersebut dalam tindakan.

Diskusi yang disarankan

Mengapa penting untuk mengatur ekspresi gen mengapa tidak mengekspresikan semua gen sepanjang waktu?

Buat daftar hipotesis dengan teman sekelas Anda tentang alasan mengapa regulasi ekspresi gen penting untuk bakteri dan archaea dan untuk eukariota. Alih-alih bakteri vs. eukariota, Anda mungkin juga ingin mempertimbangkan alasan kontras regulasi gen penting untuk organisme uniseluler versus organisme multi-seluler atau komunitas organisme uniseluler (seperti koloni bakteri).

Submasalah untuk transkripsi dan aktivitas atau RNA polimerase

Mari kita pertimbangkan gen pengkode protein dan bekerja melalui beberapa logika. Kita mulai dengan membayangkan kasus sederhana, di mana gen penyandi protein dikodekan oleh satu rangkaian DNA yang berdekatan. Kita tahu bahwa untuk menyalin gen ini, RNA polimerase perlu direkrut ke awal wilayah pengkodean. RNA polimerase tidak "pintar" sendiri. Perlu ada mekanisme, berdasarkan logika kimia, untuk membantu merekrut RNA polimerase ke awal gen pengkode protein. Demikian juga, jika proses ini akan diatur, perlu ada beberapa mekanisme, atau mekanisme, untuk menentukan kapan RNA polimerase harus direkrut ke awal gen, Kapan seharusnya tidak, dan/atau jika itu direkrut ke DNA apakah itu benar-benar harus memulai transkripsi dan berapa kali proses ini harus terjadi. Perhatikan, bahwa kalimat sebelumnya, memiliki beberapa submasalah/pertanyaan yang berbeda (misalnya kapan polimerase direkrut?; jika direkrut, haruskah memulai transkripsi?; jika memulai transkripsi, berapa kali proses ini harus diulang?). Kami juga dapat menyimpulkan, bahwa perlu ada beberapa mekanisme untuk "menginstruksikan" (lebih banyak antropomorfisme) polimerase untuk menghentikan transkripsi. Akhirnya, karena peran transkripsi adalah untuk membuat salinan RNA dari segmen genom, kita juga harus mempertimbangkan masalah/pertanyaan terkait dengan faktor lain yang mempengaruhi kelimpahan RNA, seperti mekanisme degradasi. Harus ada beberapa mekanisme dan ini kemungkinan akan terlibat dalam pengaturan proses ini.

Skema yang menunjukkan gen pengkode protein dan beberapa pertanyaan atau masalah yang perlu kita tanyakan pada diri kita sendiri atau masalah alternatif yang perlu kita ketahui solusinya jika kita ingin memahami bagaimana regulasi bagian transkripsi ekspresi gen diatur. Atribusi: Marc T. Facciotti (karya sendiri)

Aktivasi dan Represi Transkripsi

Beberapa dasar

Mari kita pertimbangkan gen pengkode protein dan bekerja melalui beberapa logika. Perlu ada beberapa mekanisme, berdasarkan logika kimia, untuk membantu merekrut RNA polimerase ke awal gen pengkode protein. Demikian juga, jika proses ini akan diatur, perlu ada beberapa mekanisme, atau mekanisme, untuk menentukan kapan RNA polimerase harus direkrut ke awal gen, kapan tidak, dan/atau direkrut ke gen. DNA, apakah benar-benar harus memulai transkripsi atau tidak dan berapa kali proses ini harus terjadi. kapan polimerase direkrut?, jika direkrut harus memulai transkripsi?, jika memulai transkripsi, berapa kali proses ini harus diulang?). Kami juga dapat menyimpulkan, bahwa perlu ada beberapa mekanisme untuk "menginstruksikan" (lebih banyak antropomorfisme) polimerase untuk menghentikan transkripsi.

Merekrut RNA polimerase ke situs tertentu

Untuk memulai transkripsi, RNA polimerase harus direkrut ke segmen DNA di dekat awal wilayah DNA yang mengkode transkrip fungsional. Fungsi RNA polimerase seperti yang dijelaskan sejauh ini, bagaimanapun, bukanlah untuk mengikat urutan tertentu melainkan untuk bergerak sepanjang setiap segmen DNA. Menemukan cara untuk merekrut polimerase ke situs tertentu karena itu tampaknya bertentangan dengan perilaku biasanya. Menjelaskan kontradiksi ini mengharuskan kita untuk memanggil sesuatu yang baru. Salah satu transkripsi dapat dimulai di mana saja dan hanya peristiwa-peristiwa yang mengarah pada transkrip produktif penuh yang melakukan sesuatu yang berguna atau sesuatu selain RNA polimerase itu sendiri membantu merekrut enzim ke awal gen. Yang terakhir, sekarang kita terima begitu saja, memang demikian.

Rekrutmen RNA polimerase dimediasi oleh protein yang disebut faktor transkripsi umum. Pada bakteri, mereka memiliki nama khusus: faktor sigma. Di archaea mereka disebut protein pengikat TATA dan faktor transkripsi IIB. Pada eukariota, kerabat protein archaeal berfungsi bersama dengan banyak orang lain untuk merekrut RNA polimerase. Faktor transkripsi umum memiliki setidaknya dua fungsi dasar: (1) Mereka mampu secara kimia mengenali urutan DNA tertentu dan (2) mereka mampu mengikat RNA polimerase. Bersama-sama kedua fungsi faktor transkripsi umum ini memecahkan masalah perekrutan enzim yang sebaliknya tidak mampu mengikat situs DNA tertentu. Dalam beberapa teks, faktor transkripsi umum (dan khususnya varietas faktor sigma) dikatakan sebagai bagian dari RNA polimerase. Sementara mereka tentu saja merupakan bagian dari kompleks ketika mereka membantu menargetkan RNA polimerase, mereka tidak melanjutkan dengan RNA polimerase setelah memulai transkripsi.

Situs DNA tempat RNA polimerase direkrut memiliki nama khusus. Ini disebut promotor. Sementara urutan DNA dari promotor yang berbeda tidak harus persis sama, urutan promotor yang berbeda biasanya memiliki beberapa sifat kimia khusus yang sama. Jelas, satu properti adalah bahwa mereka dapat berasosiasi dengan RNA polimerase. Selain itu, promotor biasanya memiliki sekuens DNA yang memfasilitasi disosiasi DNA untai ganda sehingga polimerase dapat mulai membaca dan mentranskripsikan daerah pengkode. (Catatan: secara teknis kami dapat memecah properti promotor menjadi submasalah tantangan desain. Dalam hal ini kami melewatkannya, tetapi Anda masih dapat melangkah mundur dan membuat pernyataan masalah dan atau pertanyaan yang relevan setelah Anda mengetahui tentang promotor ).

Dalam hampir semua kasus, tetapi khususnya dalam sistem eukariotik, kompleks protein yang berkumpul dengan RNA polimerase pada promotor (biasanya disebut kompleks pra-inisiasi) dapat berjumlah puluhan protein. Masing-masing protein lain ini memiliki fungsi spesifik tetapi ini terlalu banyak detail untuk menyelami Bis2A.

Model kompleks pra-inisiasi E. coli. Faktor sigma berwarna merah. DNA digambarkan sebagai tabung oranye dan struktur cincin yang berlawanan. Sisa kompleks pra-inisiasi berwarna merah muda. Perhatikan bahwa DNA memiliki daerah heliks ganda dan struktur terbuka di dalam PIC. Atribusi: Struktur diturunkan dari koordinat PDB (4YLN) Marc T. Facciotti (karya sendiri)

Model abstrak unit transkripsi generik yang ditunjukkan di atas dengan penambahan promotor dan PIC. Pertanyaan yang disebutkan sebelumnya yang kemungkinan tidak akan tercakup dalam Bis2a telah berwarna abu-abu. Facciotti (karya sendiri)

Diskusi yang disarankan

Bagaimana faktor transkripsi umum mengenali urutan spesifik DNA? Apa yang mungkin menjadi dasar kimia untuk ini? Bagaimana, sebaliknya, interaksi protein atau enzim yang berinteraksi dengan DNA secara non-spesifik berbeda? Pikirkan tentang kelompok fungsional dan ajukan hipotesis.

Negara dari promotor teregulasi

Sejak promotor merekrut RNA polimerase situs ini dan perakitan kompleks pra-inisiasi adalah situs yang jelas untuk mengatur langkah pertama ekspresi gen. Pada tingkat inisiasi transkripsi, kita sering mengklasifikasikan promotor menjadi salah satu dari tiga kelas. Yang pertama disebut pokok. Promotor konstitutif umumnya tidak diatur dengan sangat kuat. Status dasar mereka adalah "aktif". Ketika transkripsi konstitutif dari promotor sangat tinggi (relatif terhadap kebanyakan promotor lainnya), kami akan menyebut promotor itu sebagai promotor "konstitutif kuat". Sebaliknya, jika jumlah transkripsi dari promotor konstitutif rendah (relatif terhadap kebanyakan promotor lainnya), kami akan menyebut promotor itu sebagai promotor "konstitutif lemah".

Cara kedua untuk mengklasifikasikan promotor dengan menggunakan istilah diaktifkan atau setara, diinduksi. Istilah yang dapat dipertukarkan ini digunakan untuk menggambarkan promotor yang sensitif terhadap beberapa stimulus eksternal dan merespons stimulus tersebut dengan meningkatkan transkripsi. Promotor yang diaktifkan memiliki keadaan dasar umumnya menunjukkan sedikit atau tidak ada transkripsi. Transkripsi kemudian "diaktifkan" sebagai respons terhadap stimulus - stimulus menghidupkan promotor.

Akhirnya, istilah ketiga yang digunakan untuk mengklasifikasikan promotor adalah dengan menggunakan istilah tertindas. Promotor ini juga menanggapi rangsangan tetapi melakukannya dengan mengurangi transkripsi. Keadaan dasar untuk promotor ini adalah beberapa tingkat transkripsi basal dan stimulus bertindak untuk menolak atau menekan transkripsi. Transkripsi "ditekan" sebagai respons terhadap stimulus - stimulus mematikan promotor.

Contoh-contoh yang diberikan di atas mengasumsikan bahwa stimulus tunggal bertindak untuk mengatur promotor. Meskipun ini adalah kasus yang paling sederhana, banyak promotor dapat mengintegrasikan berbagai jenis informasi dan dapat diaktifkan secara bergantian oleh beberapa rangsangan dan ditekan oleh rangsangan lain.

Faktor transkripsi membantu mengatur perilaku promotor

Bagaimana promotor peka terhadap rangsangan eksternal? Secara mekanis, baik dalam aktivasi maupun represi, memerlukan protein pengatur untuk mengubah keluaran transkripsi gen yang diamati. Protein yang bertanggung jawab untuk membantu mengatur ekspresi umumnya disebut faktor transkripsi. Urutan DNA spesifik yang terikat oleh faktor transkripsi sering disebut operator dan dalam banyak kasus operator sangat dekat dengan urutan promotor.

Di sinilah nomenklatur berpotensi membingungkan - terutama ketika membandingkan penelitian bakteri dan eukariotik. Di dalam

penelitian bakteri

, jika faktor transkripsi bertindak dengan mengikat DNA dan RNA polimerase dengan cara yang meningkatkan transkripsi, maka biasanya disebut penggerak. Sebaliknya, jika faktor transkripsi bertindak dengan mengikat DNA untuk menekan atau menurunkan transkripsi gen maka disebut penekan.

Mengapa klasifikasi aktivator dan represor berpotensi bermasalah? Istilah-istilah ini, dijelaskan dalam fungsi tunggal yang diidealkan. Meskipun ini mungkin benar dalam kasus beberapa faktor transkripsi, pada kenyataannya faktor transkripsi lain dapat bertindak untuk mengaktifkan ekspresi gen dalam beberapa kondisi sementara menekan dalam kondisi lain. Beberapa faktor transkripsi hanya akan bertindak untuk memodulasi ekspresi baik naik atau turun tergantung pada konteks daripada mematikan transkripsi "off" atau mengubahnya sepenuhnya "on". Untuk menghindari beberapa kemungkinan kebingungan ini, beberapa instruktur Anda lebih memilih untuk menghindari penggunaan istilah aktivator dan represor dan sebaliknya lebih memilih untuk hanya membahas aktivitas transkripsi berbagai faktor transkripsi sebagai pengaruh positif atau negatif pada ekspresi gen dalam kasus tertentu. Jika istilah ini digunakan, Anda mungkin mendengar instruktur Anda mengatakan bahwa faktor transkripsi yang dimaksud BERTINDAK SEPERTI/SEBAGAI penekan atau BERTINDAK SEPERTI/SEBAGAI aktivator, berhati-hatilah untuk tidak menyebutnya hanya sebagai aktivator atau represor. Namun kemungkinan besar Anda akan mendengar mereka mengatakan bahwa faktor transkripsi bertindak untuk memengaruhi transkripsi secara positif atau negatif.

PERHATIAN: Tergantung pada instruktur Anda, Anda dapat membahas beberapa contoh biologis nyata dari mekanisme regulasi positif dan negatif. Contoh spesifik ini akan menggunakan nama umum dari faktor transkripsi - karena contoh biasanya diambil dari literatur bakteri, nama faktor transkripsi dapat mencakup istilah "penekan" atau "aktivator". Nama-nama ini adalah peninggalan saat pertama kali ditemukan. Cobalah untuk menghabiskan lebih banyak waktu untuk memeriksa logika cara kerja sistem daripada mencoba memasukkan ke dalam memori setiap properti khusus dari protein spesifik itu ke semua kasus lain dengan label yang sama. Artinya, hanya karena protein diberi label sebagai penekan tidak berarti protein itu secara eksklusif bertindak sebagai regulator negatif dalam semua kasus atau berinteraksi dengan sinyal eksternal dalam hal yang sama persis seperti contoh.

Diskusi yang disarankan

Menurut Anda jenis interaksi apa yang terjadi antara asam amino dari faktor transkripsi dan heliks ganda DNA? Bagaimana faktor transkripsi mengenali situs pengikatannya pada DNA?

Modulator Alosterik Protein Pengatur

Aktivitas banyak protein, termasuk protein pengatur dan berbagai faktor transkripsi, dapat dimodulasi secara alosterik berbagai faktor, termasuk oleh kelimpahan relatif molekul kecil di dalam sel. Molekul-molekul kecil ini sering disebut sebagai penginduksi atau rekan represoratau co-aktivator dan seringkali merupakan metabolit, seperti laktosa atau triptofan atau molekul pengatur kecil, seperti cAMP atau GTP. Interaksi ini memungkinkan TF untuk responsif terhadap kondisi lingkungan dan memodulasi fungsinya yang sesuai. Ini membantu untuk membuat keputusan tentang apakah akan menyalin gen atau tidak tergantung pada kelimpahan sinyal lingkungan.

Mari kita bayangkan regulator transkripsi negatif. Dalam kasus paling sederhana yang telah kami pertimbangkan sejauh ini, transkripsi gen dengan situs pengikatan untuk faktor transkripsi ini akan rendah ketika TF ada dan tinggi ketika TF tidak ada. Kita sekarang dapat menambahkan molekul kecil ke model ini. Dalam hal ini molekul kecil mampu mengikat regulator transkripsi negatif melalui set ikatan hidrogen dan ion komplementer. Dalam contoh pertama ini kita akan mempertimbangkan kasus di mana pengikatan molekul kecil ke TF menginduksi perubahan konformasi ke TF yang sangat mengurangi kemampuannya untuk mengikat DNA. Jika ini masalahnya, regulator negatif - setelah terikat oleh molekul kecilnya - akan terlepas dari DNA. Ini dengan demikian akan menghilangkan pengaruh negatif dan menyebabkan peningkatan transkripsi. Logika pengaturan ini mungkin cocok untuk berkembang dalam skenario berikut: bahan makanan molekul kecil biasanya tidak ada di lingkungan. Oleh karena itu, gen yang mengkode enzim yang akan mendegradasi/menggunakan makanan itu harus tetap "mati" sebagian besar waktu untuk melestarikan energi seluler yang akan digunakan oleh sintesisnya. Hal ini dapat dicapai dengan tindakan regulator negatif. Ketika bahan makanan muncul di lingkungan, enzim yang bertanggung jawab untuk pemrosesannya akan diekspresikan. Bahan makanan kemudian dapat bertindak dengan mengikat regulator negatif, mengubah konformasi TF, menyebabkan pelepasannya dari DNA dan mengaktifkan transkripsi enzim pemroses.

Model abstrak unit transkripsi generik yang diatur oleh regulator negatif yang aktivitasnya dimodulasi oleh molekul kecil (digambarkan oleh bintang). Dalam hal ini, pengikatan molekul kecil menyebabkan TF terlepas dari DNA. Facciotti (karya sendiri)

Kita dapat mempertimbangkan model kedua tentang bagaimana TF yang bertindak negatif dapat berinteraksi dengan molekul kecil. Dalam hal ini, TF saja tidak dapat mengikat situs regulasinya pada DNA. Namun, ketika sebuah molekul kecil mengikat TF, terjadi perubahan konformasi yang mengarahkan kembali asam amino pengikat DNA ke orientasi "benar" untuk pengikatan DNA. Kompleks molekul TF-kecil sekarang mengikat DNA dan bertindak untuk mempengaruhi transkripsi secara negatif.

Model abstrak unit transkripsi generik yang diatur oleh regulator negatif yang aktivitasnya dimodulasi oleh molekul kecil (digambarkan oleh bintang). Dalam hal ini, pengikatan molekul kecil menyebabkan TF berikatan dengan DNA. Facciotti (karya sendiri)

Perhatikan bagaimana aktivitas TF dapat dimodulasi dengan cara yang sangat berbeda oleh molekul kecil. Tergantung pada proteinnya, pengikatan sinyal eksternal ini dapat menyebabkan pengikatan kompleks molekul kecil TF ke DNA ATAU pengikatan molekul kecil dapat menyebabkan pelepasan kompleks molekul kecil TF dari DNA. Jenis contoh yang sama dapat digunakan untuk regulator positif.

Dalam kedua kasus yang diusulkan di atas, pengikatan molekul kecil ke TF akan tergantung pada seberapa kuat TF berinteraksi dengan molekul kecil. Ini akan tergantung pada jenis dan orientasi spasial gugus fungsi kimia protein, status protonasinya (jika ada), dan gugus fungsi komplementer pada molekul kecil. Oleh karena itu, tidak mengherankan untuk mengetahui bahwa pengikatan molekul kecil ke TF akan bergantung pada berbagai faktor, termasuk namun tidak terbatas pada konsentrasi relatif molekul kecil dan TF dan pH.

Apakah regulasi positif atau negatif?

Menyelesaikan titik kebingungan yang umum

Pada titik ini, tidak jarang banyak siswa Bis2a yang sedikit bingung tentang bagaimana menentukan apakah faktor transkripsi bertindak sebagai regulator positif atau negatif. Kebingungan ini sering muncul setelah diskusi tentang kemungkinan mode stimulus (yaitu molekul kecil) dapat mempengaruhi aktivitas faktor transkripsi. Ini tidak terlalu mengejutkan. Dalam contoh di atas, pengikatan molekul efektor ke faktor transkripsi dapat memiliki salah satu dari dua efek yang berbeda: (1) pengikatan molekul efektor dapat menginduksi faktor transkripsi yang terikat DNA untuk dilepaskan dari tempat pengikatannya, menekan promotor, dan "menghidupkan" ekspresi gen. (2) pengikatan molekul efektor ke faktor transkripsi dapat menginduksi TF untuk mengikat ke situs pengikatan DNA-nya, menekan transkripsi dan "mematikan" ekspresi gen. Dalam kasus pertama mungkin tampak bahwa TF bertindak untuk mengatur ekspresi secara positif, sedangkan pada contoh kedua mungkin tampak bahwa TF bertindak negatif.

Namun, dalam kedua contoh di atas, TF bertindak sebagai regulator negatif. Untuk menentukan ini kita melihat apa yang terjadi ketika TF mengikat DNA (apakah molekul kecil terikat pada TF atau tidak). Dalam kedua kasus, pengikatan TF ke DNA menekan transkripsi. Oleh karena itu TF bertindak sebagai regulator negatif. Analisis serupa dapat dilakukan dengan TF yang bertindak positif.

Perhatikan bahwa dalam beberapa kasus TF dapat bertindak sebagai regulator positif pada satu promotor dan regulator negatif pada promotor yang berbeda sehingga menggambarkan perilaku TF pada basis per kasus seringkali penting (membaca terlalu banyak dari nama yang telah ditetapkan dapat kadang menyesatkan). Protein TF lainnya dapat bertindak secara bergantian sebagai regulator positif atau negatif dari promotor yang sama tergantung pada kondisi. Sekali lagi, disarankan untuk menggambarkan perilaku TF secara khusus untuk setiap kasus.

Tes genetik untuk fungsi regulasi positif atau negatif dari TF

Bagaimana cara menentukan apakah protein pengatur berfungsi secara positif atau negatif? Tes genetik sederhana adalah menanyakan "apa yang terjadi pada ekspresi jika protein pengatur tidak ada?" Ini dapat dicapai dengan menghapus gen pengkode untuk faktor transkripsi dari genom. Jika faktor transkripsi bertindak positif, maka keberadaannya diperlukan untuk mengaktifkan transkripsi. Jika tidak ada, tidak ada protein pengatur, oleh karena itu tidak ada aktivasi, dan hasilnya adalah tingkat transkripsi yang lebih rendah dari gen target. Kebalikannya berlaku untuk faktor transkripsi yang bertindak negatif. Jika tidak ada, ekspresi harus ditingkatkan, karena gen yang menjaga ekspresi tetap rendah sudah tidak ada lagi.

Penghentian Transkripsi dan degradasi RNA

Penghentian transkripsi pada bakteri

Setelah gen ditranskripsi, polimerase bakteri perlu diinstruksikan untuk memisahkan dari template DNA dan membebaskan mRNA yang baru dibuat. Tergantung pada gen yang ditranskripsi, ada dua jenis sinyal terminasi. Satu berbasis protein dan yang lainnya berbasis RNA. Penghentian yang bergantung pada Rho dikendalikan oleh protein rho, yang melacak di belakang polimerase pada rantai mRNA yang sedang tumbuh. Menjelang akhir gen, polimerase bertemu dengan nukleotida G pada cetakan DNA dan berhenti. Akibatnya, protein rho bertabrakan dengan polimerase. Interaksi dengan rho melepaskan mRNA dari gelembung transkripsi.

pemutusan Rho-independen dikendalikan oleh urutan spesifik dalam untai cetakan DNA. Saat polimerase mendekati akhir gen yang ditranskripsi, ia bertemu dengan daerah yang kaya akan nukleotida C-G. MRNA melipat kembali dengan sendirinya, dan nukleotida C-G komplementer mengikat bersama. Hasilnya stabil jepit rambut yang menyebabkan polimerase terhenti segera setelah mulai mentranskripsi wilayah yang kaya akan nukleotida A-T. Wilayah U–A komplementer dari transkrip mRNA hanya membentuk interaksi yang lemah dengan DNA templat. Ini, ditambah dengan polimerase yang terhenti, menginduksi ketidakstabilan yang cukup bagi enzim inti untuk melepaskan diri dan membebaskan transkrip mRNA baru.

Penghentian transkripsi pada eukariota

Pengakhiran transkripsi berbeda untuk polimerase yang berbeda. Tidak seperti pada prokariota, pemanjangan oleh RNA polimerase II pada eukariota terjadi 1.000-2.000 nukleotida di luar ujung gen yang ditranskripsi. Ekor pra-mRNA ini kemudian dihilangkan dengan pembelahan selama pemrosesan mRNA. Di sisi lain, RNA polimerase I dan III membutuhkan sinyal terminasi. Gen yang ditranskripsi oleh RNA polimerase I mengandung urutan 18-nukleotida spesifik yang dikenali oleh protein terminasi. Proses penghentian pada RNA polimerase III melibatkan jepit rambut mRNA yang mirip dengan penghentian transkripsi rho-independen pada prokariota.

Penghentian transkripsi di archaea

Pengakhiran transkripsi di archaea jauh lebih sedikit dipelajari daripada di dua domain kehidupan lainnya dan masih belum dipahami dengan baik. Sementara detail fungsional cenderung menyerupai mekanisme yang telah terlihat di domain kehidupan lainnya, detailnya berada di luar cakupan kursus ini.

Degradasi RNA

Masa hidup spesies RNA yang berbeda dalam sel dapat bervariasi secara dramatis, dari detik hingga jam. Masa hidup rata-rata mRNA juga dapat bervariasi secara dramatis tergantung pada organisme. Misalnya, masa hidup rata-rata untuk mRNA di E. coli adalah ~ 5 menit. Waktu paruh mRNA dalam ragi adalah ~ 20 menit dan 600 menit untuk sel manusia. Beberapa degradasi "ditargetkan". Artinya, beberapa transkrip menyertakan urutan pendek yang menargetkan mereka untuk enzim pendegradasi RNA, mempercepat laju degradasi. Tidak perlu terlalu banyak imajinasi untuk menyimpulkan bahwa proses ini mungkin juga disetel secara evolusioner untuk gen yang berbeda. Cukup menyadari bahwa degradasi - dan pengaturan degradasi - juga dapat menjadi faktor dalam mengendalikan ekspresi gen yang cukup untuk Bis2a.

Penghentian penyetelan

Seperti semua proses biologis lainnya, penghentian transkripsi tidak sempurna. Kadang-kadang, RNA polimerase mampu membaca urutan terminator dan menyalin gen yang berdekatan. Hal ini terutama berlaku pada genom bakteri dan archaeal di mana kepadatan gen pengkode tinggi. Pembacaan transkripsi ini dapat memiliki berbagai efek tetapi dua hasil yang paling umum adalah: (1) Jika gen yang berdekatan dikodekan pada untai DNA yang sama dengan gen yang ditranskripsi secara aktif, gen yang berdekatan juga dapat ditranskripsi. (2) Jika gen yang berdekatan ditranskripsikan pada untai berlawanan dari gen yang ditranskripsi secara aktif, RNA polimerase membaca-melalui dengan mengganggu polimerase yang secara aktif mentranskripsi gen tetangga. Tidak mengherankan, dalam beberapa kasus biologi telah mengembangkan mekanisme yang bertindak untuk meminimalkan pengaruh pembacaan dan untuk mengambil keuntungan darinya. Oleh karena itu, "kekuatan" terminator - dan keefektifannya dalam menghentikan transkripsi ke arah tertentu - "disetel" oleh Alam dan "digunakan" (perhatikan antropomorfisme dalam tanda kutip) untuk mengatur ekspresi gen.

Model abstrak dari unit transkripsi dasar lengkap dan berbagai "bagian" yang dikodekan pada DNA yang dapat mempengaruhi ekspresi gen. Kami berharap siswa di Bis2A dapat menciptakan kembali sosok konseptual serupa dari ingatan. Facciotti (karya sendiri)

Ringkasan regulasi gen

Dalam teks sebelumnya kami telah memeriksa beberapa cara untuk mulai memecahkan beberapa tantangan desain yang terkait dengan mengatur jumlah transkrip yang dibuat untuk satu wilayah pengkodean genom. Kami telah melihat secara abstrak beberapa proses yang bertanggung jawab untuk mengendalikan inisiasi transkripsi, bagaimana ini dapat dibuat sensitif terhadap faktor lingkungan, dan secara singkat pada proses yang mengakhiri transkripsi dan menangani degradasi aktif RNA. Kami telah menghindari lebih banyak Setiap proses ini dapat secara kuantitatif disetel oleh alam menjadi "lebih kuat" atau "lebih lemah". Penting untuk disadari bahwa nilai sebenarnya dari "kekuatan" (misalnya kekuatan promotor, tingkat degradasi, dll.) mempengaruhi perilaku proses keseluruhan dengan cara yang berpotensi penting secara fungsional.

Contoh Regulasi Gen Bakteri

Bagian ini menjelaskan dua contoh regulasi transkripsi pada bakteri. Ini disajikan sebagai contoh ilustrasi. Gunakan contoh-contoh ini untuk mempelajari beberapa prinsip dasar tentang mekanisme regulasi transkripsi. Waspadalah di kelas, dalam diskusi, dan dalam panduan belajar untuk perluasan ide-ide ini dan gunakan ini untuk menjelaskan mekanisme pengaturan yang digunakan untuk mengatur gen lain.

Contoh Regulasi Gen di E. coli

DNA bakteri dan archaea biasanya diatur menjadi satu atau lebih kromosom melingkar di sitoplasma. Agregat padat DNA yang dapat dilihat dalam mikrograf elektron disebut nukleoid. Pada bakteri dan archaea, gen, yang ekspresinya perlu dikoordinasikan secara ketat (misalnya gen yang mengkode protein yang terlibat dalam jalur biokimia yang sama) sering dikelompokkan bersama dalam genom. Ketika ekspresi beberapa gen dikendalikan oleh promotor yang sama dan satu transkrip diproduksi, unit ekspresi ini disebut operon. Misalnya pada bakteri Escherschia coli semua gen yang dibutuhkan untuk memanfaatkan laktosa dikodekan di samping satu sama lain dalam genom. Susunan ini disebut laktosa (atau lac) operasi. Hal ini sering terjadi pada bakteri dan archaea bahwa hampir 50% dari semua gen dikodekan menjadi operon dari dua atau lebih gen.

Peran Promotor

The first level of control of gene expression is at the promoter itself. Some promoters recruit RNA polymerase and turn those DNA-protein binding events into transcripts more efficiently than other promoters. This intrinsic property of a promoter, it's ability to produce transcript at a particular rate, is referred to as promoter strength. The stronger the promoter, the more RNA is made in any given time period. Promoter strength can be "tuned" by Nature in very small or very large steps by changing the nucleotide sequence the promoter (e.g. mutating the promoter). This results in families of promoters with different strengths that can be used to control the maximum rate of gene expression for certain genes.

UC Davis Undergraduate Connection:

A group of UC Davis students interested in synthetic biology used this idea to create synthetic promoter libraries for engineering microbes as part of their design project for the 2011 iGEM competition.

Example #1: Trp Operon

Logic for regulating tryptophan biosynthesis

E. coli, like all organisms, needs to either synthesize or consume amino acids to survive. The amino acid tryptophan is one such amino acid. E. colican either import tryptophan from the environment (eating what it can scavenge from the world around it) or synthesize tryptophan de novo using enzymes that are encoded by five genes. These five genes are encoded next to each other in the E. coli genome into what is called the tryptophan (trp) operon (Figure below). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when environmental tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized. See the figure and paragraphs below for a mechanistic explanation.

Organization of the trp operon

Five genomic regions encoding tryptophan biosynthesis enzymes are arranged sequentially on the chromosome and are under the control of a single promoter - they are organized into an operon. Just before the coding region is the transcriptional start site. This is, as the name implies, the location where the RNA polymerase starts a new transcript. The promoter sequence is further upstream of the transcriptional start site.

A DNA sequence called an "operator" is also encoded between the promoter and the first trp coding gene. Ini operator is the DNA sequence to which the transcription factor protein will bind.

A few more details regarding TF binding sites

It should be noted that the use of the term "operator" is limited to just a few regulatory systems and almost always refers to the binding site for a negatively acting transcription factor. Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory proteins allowing them to perform their appropriate function (e.g. repress or activate transcription). This theme will be repeated universally across biology whether the "operator" term is used or not.

Moreover, while the specific examples you will be show depict TF binding sites in their known locations, these locations are not universal to all systems. Transcription factor binding sites can vary in location relative to the promoter. There are some patterns (e.g. positive regulators are often upstream of the promoter and negative regulators bind downstream), but these generalizations are not true for all cases. Again, the key thing to remember is that transcription factors (both positive and negatively acting) have binding sites with which they interact to help regulate the initiation of transcription by RNA polymerase.

The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind to the transcription factor and allow the TF-tryptophan complex to bind at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes. When tryptophan is absent, the transcription factor does not bind to the operator and the genes are transcribed.
Attribution: Marc T. Facciotti (own work)

Regulation of the trp operon

When tryptophan is present in the cell: two tryptophan molecules bind to the trp repressor protein. When tryptophan binds to the transcription factor it causes a conformational change in the protein which now allows the TF-tryptophan complex to bind to the trp operator sequence. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes. When tryptophan is not present in the cell, the transcription factor does not bind to the operator; therefore, the transcription proceeds, the tryptophan utilization genes are transcribed and translated, and tryptophan is thus synthesized.

Since the transcription factor actively binds to the operator to keep the genes turned off, the trp operon is said to be "negatively regulated" and the proteins that bind to the operator to silence trp expression are negative regulators.

Suggested discussion

Do you think that the constitutive expression levels of the trp operon are high or low? Mengapa?

Suggestion discussion

Suppose nature took a different approach to regulating the trp operon. Design a method for regulating the expression of the trp operon with a positive regulator instead of a negative regulator. (hint: we ask this kind of question all of the time on exams)

External link

Watch this video to learn more about the trp operasi.

Example #2: The lac operon

Rationale for studying the lac operon

In this example, we examine the regulation of genes encoding proteins whose physiological role is to import and assimilate the disaccharide lactose, the lac operasi. The story of the regulation of lac operon is a common example used in many introductory biology classes to illustrate basic principles of inducible gene regulation. We choose to describe this example second because it is, in our estimation, more complicated than the previous example involving the activity of a single negatively acting transcription factor. By contrast, the regulation of the lac operon is, in our opinion, a wonderful example of how the coordinated activity of both positive and negative regulators around the same promoter can be used to integrate multiple different sources of cellular information to regulate the expression of genes.

As you go through this example, keep in mind the last point. For many Bis2a instructors it is more important for you to learn the lac operon story and guiding principles than it is for you to memorize the logic table presented below. When this is the case, the instructor will usually make a point to let you know. These instructors often deliberately do NOT include exam questions about the lac operasi. Rather they will test you on whether you understood the fundamental principles underlying the regulatory mechanisms that you study using the lac operon example. If it's not clear what the instructor wants you should ask.

The utilization of lactose

Lactose is a disaccharide composed of the hexoses glucose and galactose. It is commonly found in high abundance in milk and some milk products. As one can imagine, the disaccharide can be an important food-stuff for microbes that are able to utilize its two hexoses. coli is able to use multiple different sugars as energy and carbon sources, including lactose and the lac operon is a structure that encodes the genes necessary to acquire and process lactose from the local environment. Lactose, however, has not been frequently encountered by E. coli during its evolution and therefore the genes of the lac operon must typically be repressed (i.e. "turned off") when lactose is absent. Driving transcription of these genes when lactose is absent would waste precious cellular energy. By contrast, when lactose is present, it would make logical sense for the genes responsible for the utilization of the sugar to be expressed (i.e. "turned on"). So far the story is very similar to that of the tryptophan operon described above.

However, there is a catch. Experiments conducted in the 1950's by Jacob and Monod clearly demonstrated that E. coli prefers to utilize all the glucose present in the environment before it begins to utilize lactose. This means that the mechanism used to decide whether or not to express the lactose utilization genes must be able to integrate two types of information (1) the concentration of glucose and (2) the concentration of lactose. While this could theoretically be accomplished in multiple ways, we will examine how the lac operon accomplishes this by using multiple transcription factors.

The transcriptional regulators of the lac operon

NS lac repressor - a direct sensor of lactose

As noted, the lac operon normally has very low to no transcriptional output in the absence of lactose. This is due to two factors: (1) the constitutive promoter strength for the operon is relatively low and (2) the constant presence of the LacI repressor protein negatively influences transcription. This protein binds to the operator site near the promoter and blocks RNA polymerase from transcribing the lac operon genes. By contrast, if lactose is present, lactose will bind to the LacI protein, inducing a conformational change that prevents LacI-lactose complex from binding to its binding sites. Therefore, when lactose is present the negative regulatory LacI is not bound to the its binding site and transcription of lactose utilizing genes can proceed.

CAP protein - an indirect sensor of glucose

Di dalam E. coli, when glucose levels drop, the small molecule cyclic AMP (cAMP) begins to accumulate in the cell. cAMP is a common signaling molecule that is involved in glucose and energy metabolism in many organisms. When glucose levels decline in the cell, the increasing concentrations of cAMP allow this compound to bind to the positive transcriptional regulator called catabolite activator protein (CAP) - also referred to as CRP. cAMP-CAP complex has many sites located throughout the E. coli genome and many of these sites are located near the promoters of many operons that control the processing of various sugars.

Dalam lac operon, the cAMP-CAP binding site is located upstream of the promoter. Binding of cAMP-CAP to the DNA helps to recruit and retain RNA polymerase to the promoter. The increased occupancy of RNA polymerase to its promoter, in turn, results in increased transcriptional output. In this case the CAP protein is acting as a positive regulator.

Note that the CAP-cAMP complex can, in other operons, also act as a negative regulator depending upon where the binding site for CAP-cAMP complex is located relative to the RNA polymerase binding site.

Putting it all together: Inducing expression of the lac operon

For the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed. When this condition is achieved the LacI-lactose complex dissociates the negative regulator from near the promoter, freeing the RNA polymerase to transcribe the operon's genes. Moreover, high cAMP (indirectly indicative of low glucose) levels trigger the formation of the CAP-cAMP complex. This TF-inducer pair now bind near the promoter and act to positively recruit the RNA polymerase. This added positive influence boosts transcriptional output and lactose can be efficiently utilized. The mechanistic output of other combinations of binary glucose and lactose conditions are descried in the table below and in the figure that follows.

Truth Table for Lac Operon

Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source.
Attribution: Marc T. Facciotti (own work)
Signals that Induce or Repress Transcription of the lac operon
GlucoseCAP bindsLactoseRepressor bindsTranscription
+--+Tidak
+-+-Beberapa
-+-+Tidak
-++-Ya

A more nuanced view of lac repressor function

The description of the lac repressor's function correctly describes the logic of the control mechanism used around the lac promoter. However, the molecular description of binding sites is a bit overly simplified. In reality the lac repressor has three similar, but not identical, binding sites called Operator 1, Operator 2, and Operator 3. Operator 1 is very close to the transcript start site (denoted +1). Operator 2 is located about +400nt into the coding region of the LacZ protein. Operator 3 is located about -80nt before the transcript start site (just "outside" of the CAP binding site).

The lac operon regulatory region depicting the promoter, three lac operators, and CAP binding site. The coding region for the Lac Z protein is also shown relative to the operator sequences. Note that two of the operators are in the protein coding region - there are multiple different types of information simultaneously encoded in the DNA.
Attribution: Marc T. Facciotti (own work)

The lac repressor tetramer (blue) depicted binding two operators on a strand of looped DNA (orange).
Attribution: Marc T. Facciotti (own work) - Adapted from Goodsell (https://pdb101.rcsb.org/motm/39)

Eukaryotic Gene Regulation

Regulation overview

As was previously noted, regulation is all about decision making. Gene regulation, as a general topic, is related to making decisions about the functional expression of genetic material. Whether the final product is an RNA species or a protein, the production of the final expressed product requires processes that take multiple steps. We have spent some time discussing some of these steps (i.e. transcription and translation) and some of the mechanisms that nature uses for sensing cellular and environmental information to regulate the initiation of transcription.

When we discussed the concept of strong and weak promoters we introduced the idea that regulating the amount (number of molecules) of transcript that was produced from a promoter in some unit of time might also be important for function. This should not be entirely surprising. For a protein coding gene, the more transcript that is produced, the greater potential there is to make more protein. This might be important in cases where making a lot of a particular enzyme is key for survival. By contrast, in other cases only a little protein is required and making too much would be a waste of cellular resources. In this case low levels of transcription might be preferred. Promoters of differing strengths can accommodate these varying needs. With regards to transcript number, we also briefly mentioned that synthesis is not the only way to regulate abundance. Degradation processes are also important to consider.

In this section, we add to these themes by focusing on eukaryotic regulatory processes. Specifically, we examine - and sometimes re-examine - some of the multiple steps that are required to express genetic material in eukaryotic organisms in the context of regulation. We want you not only to think about the processes but also to recognize that each step in the process of expression is also an opportunity to fine tune not only the abundance of a transcript or protein but also its functional state, form (or variant), and/or stability. Each of these additional factors may also be vitally important to consider for influencing the abundance of conditionally-specific functional variants.

Structural differences between bacterial and eukaryotic cells influencing gene regulation

The defining hallmark of the eukaryotic cell is the nucleus, a double membrane that encloses the cell's hereditary material. In order to efficiently fits the organism's DNA into the confined space of the nucleus, the DNA is first packaged and organized by protein into a structure called chromatin. This packaging of the nuclear material reduces access to specific parts of the chromatin. Indeed, some elements of the DNA are so tightly packed that the transcriptional machinery cannot access regulatory sites like promoters. This means that one of the first sites of transcriptional regulation in eukaryotes must be the control access to the DNA itself. Chromatin proteins can be subject to enzymatic modification that can influence whether they bind tightly (limited transcriptional access) or more loosely (greater transcriptional access) to a segment of DNA . This process of modification - whichever direction is considered first - is reversible. Therefore DNA can be dynamically sequestered and made available when the "time is right".

The regulation of gene expression in eukaryotes also involves some of the same additional fundamental mechanisms discussed in the module on bacterial regulation (i.e. the use of strong or weak promoters, transcription factors, terminators etc.) but the actual number of proteins involved is typically much greater in eukaryotes than bacteria or archaea.

The post-transcriptional enzymatic processing of RNA that occurs in the nucleus and the export of the mature mRNA to the cytosol are two additional difference between bacterial and eukaryotic gene regulation. We will consider this level of regulation in more detail below.

Depiction of some key differences between the processes of bacterial and eukaryotic gene expression. Note in this case the presence of histone and histone modifiers, the splicing of pre-mRNA, and the export of the mature RNA from the nucleus as key differentiators between the bacterial and eukaryotic systems.
Attribution: Marc T. Facciotti (own work)

DNA Packing and Epigenetic Markers

The DNA in eukaryotic cells is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments of the chromosomes can be easily accessed as needed by the cell. Areas of the chromosomes that are more tightly compacted will be harder for proteins to bind and therefore lead to reduced gene expression of genes encoded in those areas. Regions of the genome that are loosely compacted will be easier for proteins to access, thus increasing the likelihood that the gene will be transcribed. Discussed here are the ways in which cells regulate the density of DNA compaction.

DNA packing

The first level of organization, or packing, is the winding of DNA strands around histone protein. Histones package and order DNA into structural units called nucleosomes, which can control the access of proteins to specific DNA regions. Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string. These beads (nucleosome complexes) can move along the string (DNA) to alter which areas of the DNA are accessible to transcriptional machinery. While nucleosomes can move to open the chromosome structure to expose a segment of DNA, they do so in a very controlled manner.

DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

Histone Modification

How the histone proteins move is dependent on chemical signals found on both the histone proteins and on the DNA. These chemical signals are chemical tags added to histone proteins and the DNA that tell the histones if a chromosomal region should be "open" or "closed". The figure below depicts modifications to histone proteins and DNA. These tags are not permanent, but may be added or removed as needed. They are chemical modifications (phosphate, methyl, or acetyl groups) that are attached to specific amino acids in the histone proteins or to the nucleotides of the DNA. The tags do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins. DNA is a negatively charged molecule; therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a large positive charge; by adding chemical modifications like acetyl groups, the charge becomes less positive.

Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

Suggested discussion

Why do histone proteins normally have a large amount of positive charges (histones contain a high number of lysine amino acids). Would removal of the positive charges cause a tightening of loosening of the histone-DNA interaction?

Suggested discussion

Predict the state of the histones in areas of the genome that are transcribed regularly. How do these differ from areas that do not experience high levels of transcription?

DNA Modification

The DNA molecule itself can also be modified. This occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) often found in the promoter regions of genes. When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added). This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.

Epigenetic changes do not result in permanent changes in the DNA sequence. Epigenetic changes alter the chromatin structure (protein-DNA complex) to allow or deny access to transcribe genes. DNA modification such as methylation on cytosine nucleotides can either recruit repressor proteins that block RNA polymerase's access to transcribe a gene or they can aid in compacting the DNA to block all protein access to that area of the genome. These changes are reversible whereas mutations are not, however, epigenetic changes to the chromosome can also be inherited.
Source: modified from https://researcherblogski.wordpress....r/dudiwarsito/

Regulation of gene expression through chromatin remodeling is called epigenetic regulation. Epigenetic means “around genetics.” The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent. Instead, these changes are temporary (although they often persist through multiple rounds of cell division and can be inherited) and alter the chromosomal structure (open or closed) as needed.

External link

View this video that describes how epigenetic regulation controls gene expression.

Eukaryotic gene structure and RNA processing

Eukaryotic gene structure

Many eukaryotic genes, particularly those encoding protein products, are encoded on the genome discontinuously. That is, the coding region is broken into pieces by intervening non-coding gene elements. The coding regions are termed ekson while the intervening non-coding elements are termed intron. The figure below depicts a generic eukaryotic gene.

The parts of a typical discontinuous eukaryotic gene. Facciotti (own work)

Parts of a generic eukaryotic gene include familiar elements like a promoter and terminator. Between those two elements, the region encoding all of the elements of the gene that have the potential to be translated (they have no stop codons), like in bacterial systems, is called the open reading frame (ORF). Enhancer and/or silencer elements are regions of the DNA that serve to recruit regulatory proteins. These can be relatively close to the promoter, like in bacterial systems, or thousands of nucleotides away. Also present in many bacterial transcripts, 5' and 3' untranslated regions (UTRs) also exist. These regions of the gene encode segments of the transcript, which, as their names imply, are not translated and sit 5' and 3', respectively, to the ORF. The UTRs typically encode some regulatory elements critical for regulating transcription or steps of gene expression that occur post-transcriptionally.

The RNA species resulting from the transcription of these genes are also discontinuous and must therefore be processed before exiting the nucleus to be translated or used in the cytosol as mature RNAs. In eukaryotic systems this includes RNA splicing, 5' capping, 3' end cleavage and polyadenylation. This series of steps is a complex molecular process that must occur within the closed confines of the nucleus. Each one of these steps provides an opportunity for regulating the abundance of exported transcripts and the functional forms that these transcripts will take. While these would be topics for more advanced courses, think about how to frame some of the following topics as subproblems of the Design Challenge of genetic regulation. If nothing else, begin to appreciate the highly orchestrated molecular dance that must occur to express a gene and how this is a stunning bit of evolutionary engineering.

5' capping

Like in bacterial systems, eukaryotic systems must assemble a pre-initiation complex at and around the promoter sequence to initiate transcription. The complexes that assemble in eukaryotes serve many of the same function as those in bacterial systems but they are significantly more complex, involving many more regulatory proteins. This added complexity allows for a greater degree of regulation and for the assembly of proteins with functions that occur predominantly in eukaryotic systems. One of these additional functions is the "capping" of nascent transcripts.

In eukaryotic protein coding genes, the RNA that is first produced is called the pre-mRNA. The "pre" prefix signifies that this is not the full mature mRNA that will be translated and that it first requires some processing. The modification known as 5'-capping occurs after the pre-mRNA is about 20-30 nucleotides in length. At this point the pre-RNA typically receives its first post-transcriptional modification, a 5'-cap. The "cap" is a chemical modification - a 7-methylguanosine - whose addition to the 5' end of the transcript is enzymatically catalyzed by multiple enzymes called the capping enzyme complex (CEC) a group of multiple enzymes that carry out sequential steps involved in adding the 5'-cap. The CEC binds to the RNA polymerase very early in transcription and carries out a modification of the 5' triphosphate, the subsequent transfer of at GTP to this end (connecting the two nucleotides using a unique 5'-to-5' linkage), the methylation of the newly transferred guanine, and in some transcripts the additional modifications to the first few nucleotides. This 5'-cap appears to function by protecting the emerging transcript from degradation and is quickly bound by RNA binding proteins known as the cap-binding complex (CBC). There is some evidence that this modification and the proteins bound to it play a role in targeting the transcript for export from the nucleus. Protecting the nascent RNA from degradation is not only important for conserving the energy invested in creating the transcript but is clearly involved in regulating the abundance of fully-functional transcript that is produced. Moreover, the role of the 5'-cap in guiding the transcript for export will directly help to regulate not only the amount of transcript that is made but, perhaps more importantly, the amount of transcript that is exported to the cytoplasm that has the potential to be translated.

The structure of a typical 7-methylguanylate cap. Facciotti (own work)

Transcript splicing

Nascent transcripts must be processed into mature RNAs by joining exons and removing the intervening introns. This is accomplished by a multicomponent complex of RNA and proteins called the spliceosome. The spliceosome complex assembles on the nascent transcript and in many cases the decisions about which introns to combine into a mature transcript are made at this point. How these decisions are made is still not completely understood but involves the recognition of specific DNA sequences at the splice sites by RNA and protein species and several catalytic events. It is interesting to note that the catalytic portion of the spliceosome is made of RNA rather than protein. Recall that the ribosome is another example of a RNA-protein complex where the RNA serves as the primary catalytic component. The selection of which splice variant to make is a form of regulating gene expression. In this case rather than simply influencing abundance of a transcript, alternative splicing allows the cell to make decisions about which form of transcript is made.

The alternative splice forms of genes that result in protein products of related structure but of varying function are known as isoforms. The creation of isoforms is common in eukaryotic systems and is known to be important in different stages of development in multicellular organisms and in defining the functions of different cell types. By encoding multiple possible gene products from a single gene whose transcription initiation is encoded from a single transcriptional regulatory site (by making the decision of which end-product to produce post-transcriptionally) obviates the need to create and maintain independent copies of each gene in different parts of the genome and evolving independent regulatory sites. Therefore, the ability to form multiple isoforms from a single coding region is though to be evolutionarily advantageous because it enables some efficiency in DNA coding, minimizes transcriptional regulatory complexity, and may lower the energy burden of maintaining more DNA and protecting it from mutation. Some examples of possible outcomes of alternative splicing can include: the generation of enzyme variants with differential substrate affinity or catalytic rates; signal sequences that target proteins to various sub-cellular compartments can be changed; entirely new functions, via the swapping of protein domains can be created. These are just a few examples.

One additional interesting possible outcome of alternative splicing is the introduction of stop codons that can, through a mechanism that seems to require translation, lead to the targeted decay of the transcript. This means that, in addition to the control of transcription initiation and 5'-capping, alternative splicing can also be considered one of the regulatory mechanisms that may influence transcript abundance. The effects of alternative splicing are therefore potentially broad - from complete loss of function to novel and diversified function to regulatory effects.

A figure depicting some of the different modes of alternative splicing illustrating how different splice variants can lead to different protein forms.
Attribution: Marc T. Facciotti (own work)

3' end cleavage and polyadenylation

One final modification is made to nascent pre-mRNAs before they leave the nucleus - the cleavage of the 3' end and its polyadenylation. This two step process is catalyzed by two different enzymes (as depicted below) and may decorate the 3' end of transcripts with up to nearly 200 nucleotides. This modification enhances the stability of the transcript. Generally, the more As in the polyA tag the longer lifetime that transcript has. The polyA tag also seems to play a role in the export of the transcript from the nucleus. Therefore, the 3' polyA tag plays a role in gene expression by regulating functional transcript abundance and how much is exported from the nucleus for translation.

A two step process is involved in modifying the 3' ends of transcripts prior to nuclear exports. These include cutting transcripts just downstream of a conserved sequence (AAUAAA) and transferring adenylate groups. Both processes are enzymatically catalyzed.
Attribution: Marc T. Facciotti (own work)

MikroRNA

RNA Stability and microRNAs

In addition to the modifications of the pre-RNA described above and the associated proteins that bind to the nascent and transcripts, there are other factors that can influence the stability of the RNA in the cell. One example are elements called microRNAs. The microRNAs, or miRNAs, are short RNA molecules that are only 21–24 nucleotides in length. The miRNAs are transcribed in the nucleus as longer pre-miRNAs. These pre-miRNAs are subsequently chopped into mature miRNAs by a protein called dicer. These mature miRNAs recognize a specific sequence of a target RNA through complementary base pairing. miRNAs, however, also associate with a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). RISC binds a target mRNA, along with the miRNA, to degrade the target mRNA. Together, miRNAs and the RISC complex rapidly destroy the RNA molecule. As one might expect, the transcription of pre-miRNAs and their subsequent processing is also tightly regulated.

Nuclear export

Nuclear export

Fully processed, mature transcripts, must be exported through the nucleus. Not surprisingly this process involves the coordination of a mature RNA species to which are bound many accessory proteins - some of which have been intimately involved in the modifications discussed above - and a protein complex called the nuclear pore complex (NPC). Transport through the NPC allows flow of proteins and RNA species to move in both directions and is mediated by a number of proteins. This process can be used to selectively regulate the transport of various transcripts depending on which proteins associate with the transcript in question. This means that not all transcripts are treated equally by the NPC - depending on modification state and the proteins that have associated with a specific species of RNA it can be moved either more or less efficiently across the nuclear membrane. Since the rate of movement across the pore will influence the abundance of mature transcript that is exported into the cytosol for translation export control is another example of a step in the process of gene regulation that can be modulated. In addition, recent research has implicated interactions between the NPC and transcription factors in the regulation of transcription initiation, likely through some mechanism whereby the transcription factors tether themselves to the nuclear pores. This last example demonstrates how interconnected the regulation of gene expression is across the multiple steps of this complex process.

Many additional details of the processes described above are known to some level of detail, but many more questions remain to be answered. For the sake of Bis2a it is sufficient to begin forming a model of the steps that occur in the production of a mature transcript in eukaryotic organisms. We have painted a picture with very broad strokes, trying to present a scene that reflect what happens generally in all eukaryotes. In addition to learning the key differentiating features of eukaryotic gene regulation, we would also like for Bis2a students to begin thinking of each of these steps as an opportunity for Nature to regulate gene expression in some way and to be able to rationalize how deficiencies or changes in these pathways - potentially introduced through mutation - might influence gene expression.

While we did not explicitly bring up the Design Challenge or Energy Story here these formalisms are equally adept at helping you to make some sense of what is being described. We encourage you to try making an Energy Story for various processes. We also encourage you to use the Design Challenge rubric to reexamine the stories above: identify problems that need solving; hypothesize potential solutions and criteria for success. Use there formalisms to dig deeper and ask new questions/identify new problems or things that you don't know about the processes is what experts do. Chances are that doing this suggested exercise will lead you to identify a direction of research that someone has already pursued (you'll feel pretty smart about that!). Alternatively, you may raise some brand new question that no one has thought of yet.

Control of Protein Abundance

After an mRNA has been transported to the cytoplasm, it is translated into protein. Control of this process is largely dependent on the RNA molecule. As previously discussed, the stability of the RNA will have a large impact on its translation into a protein. As the stability changes, the amount of time that it is available for translation also changes.

The initiation complex and translation rate

Like transcription, translation is controlled by proteins complexes of proteins and nucleic acids that must associate to initiate the process. In translation, one of the first complexes that must assembles to start the process is referred to as the initiation complex. The first protein to bind to the mRNA that helps initiate translation is called eukaryotic initiation factor-2 (eIF-2). Activity of the eIF-2 protein is controlled by multiple factors. The first is whether or not it is bound to a molecule of GTP. When the eIF-2 is bound to GTP it is considered to be in an active form. The eIF-2 protein bound to GTP can bind to the small 40S ribosomal subunit. When bound, the eIF-2/40S ribosome complex, bringing with it the mRNA to be translated, also recruits the methionine initiator tRNA associates. At this point, when the initiator complex is assembled, the GTP is hydrolyzed into GDP creating an "inactive form of eIF-2 that is released, along with the inorganic phosphate, from the complex. This step, in turn, allows the large 60S ribosomal subunit to bind and to begin translating the RNA. The binding of eIF-2 to the RNA further controlled by protein phosphorylation. When eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP thus inhibiting the initiation complex from forming - translation is therefore inhibited (see the figure below). In the dephosphorylated state eIF-2 can bind GTP and allow the assembly of the translation initiation complex as described above. The ability of the cell therefore to tune the assembly of the translation invitation complex via a reversible chemical modification (phosphorylation) to a regulatory protein is another example of how Nature has taken advantage of even this seemingly simple step to tuned gene expression.

An increase in phosphorylation levels of eIF-2 has been observed in patients with neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s. What impact do you think this might have on protein synthesis?

Chemical Modifications, Protein Activity, and Longevity

Not to be outdone by nucleic acids, proteins can also be chemically modified with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins can regulate their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell—for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.

Chemical modifications can occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. In addition to regulating the function of the proteins themselves, if these changes occur on specific proteins they can alter epigenetic accessibility (in the case of histone modification), transcription (transcription factors), mRNA stability (RNA binding proteins), or translation (eIF-2) thus feeding back and regulating various parts of the process of gene expression. In the case of modification to regulatory proteins, this can be an efficient way for the cell to rapidly change the levels of specific proteins in response to the environment by regulating various steps in the process.

The addition of an ubiquitin group has another function - it marks that protein for degradation. Ubiquitin is a small molecule that acts like a flag indicating that the tagged proteins should be targeted to an organelle called the proteasome. This organelle is a large multi-protein complex that functions to cleave proteins into smaller pieces that can then be recycled. Ubiquitination (the addition of a ubiquitin tag), therefore helps to control gene expression by altering the functional lifetime of the protein product.

Proteins with ubiquitin tags are marked for degradation within the proteasome.

In conclusion, we see that gene regulation is complex and that it can be modulated at each step in the process of expressing a functional gene product. Moreover, the regulatory elements that happen at each step can act to influence other regulatory steps both earlier and later in the process of gene expression (i.e. the process of chemically altering a transcription factor can influence the regulation of its own transcription many steps earlier in the process). These complex sets of interactions form what are known as gene regulatory networks. Understanding the structure and dynamics of these networks is critical for understanding how different cells function, the basis for numerous diseases, developmental processes, and how cells make decisions about how to react to the many factors that are in constant flux both inside and outside.


Tonton videonya: Killiney Hill Park - IRLANDIA (Oktober 2022).