Lecture Molecular biology (Fifth Edition): Chapter 11 - Robert F. Weaver

Chia sẻ: Nnmm Nnmm | Ngày: | Loại File: PPT | Số trang:38

Thêm vào BST

Báo xấu

81
lượt xem 3
download

Download Vui lòng tải xuống để xem tài liệu đầy đủ

Chapter 11 - general transcription factors in eukaryotes. Eukaryotic RNA polymerases, unlike their bacterial counterparts, are incapable of binding by themselves to their respective promoters. Instead, they rely on proteins called transcription factors to show them the way. Such factors are grouped into two classes: general transcription factors and gene-specifi c transcription factors (activators). In this chapter we will survey the general transcription factors that interact with all three RNA polymerases and their promoters.

Chủ đề:

Bình luận(0) Đăng nhập để gửi bình luận!

Lưu

Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 11 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 11 General Transcription Factors in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transcription in Eukaryotes • Eukaryotic RNA polymerases, unlike their bacterial counterparts, are incapable of binding by themselves to their respective promoters • Eukaryotic RNA polymerases rely on proteins called transcription factors to show them the way • Two classes: general transcription factors and gene-specific transcription factors (activators) 11-2
11.1 Class II Factors • General transcription factors combine with RNA polymerase to form a preinitiation complex – This complex is able to initiate transcription when nucleotides are available – Tight binding involves formation of an open promoter complex with DNA at the transcription start site that has melted • The assembly of preinitiation complexes involving polymerase II is quite complex 11-3
The Class II Preinitiation Complex • Class II preinitiation complex contains: – RNA Polymerase II – 6 general transcription factors: • TFIIA, TFIIB, TFIID, TFIIE, and TFIIH • The transcription factors (TF) and polymerase bind the preinitiation complex in a specific order (as studied in vitro) 11-4
Four Distinct Preinitiation Complexes • Transcription factors bind to class II promoters in the following order in vitro: • TFIID with help from TFIIA binds to the TATA box forming the DA complex • TFIIB binds next generating the DAB complex • TFIIF helps RNA polymerase bind to a region from -34 to +17, now it is DABPolF complex • Last the TFIIE then TFIIH bind to form the complete preinitiation complex = DABPolFEH • In vitro, the participation of TFIIA seems to be optional 11-5
Model of Formation of the DABPolF Complex 11-6
Structure and Function of TFIID TFIID contains several subunits – TATA-box binding protein (TBP) • Highly evolutionarily conserved • Binds to the minor groove of the TATA box – Saddle-shaped TBP lines up with DNA – Underside of the saddle forces open the minor groove – The TATA box is bent into 80° curve – TBP-associated factors (TAFs) specific for class II 11-7
Structure of the TBP-TATA box complex 11-8
The Versatility of TBP • Genetic studies have demonstrated TBP mutant cell extracts are deficient in: – Transcription of class II genes – Transcription of class I and III genes • TBP is a universal transcription factor required by all three classes of genes • Required in transcription of at least some genes of Archaea, single-celled organisms lacking nuclei 11-9
The TBP-Associated Factors • These are also called TAFs (TAFIIs is written to denote transcription of class II genes) • 13 TAFs have been identified and associated with class II preinitiation complexes • The core TAFs were first named according to their molecular mass but have now been renamed according to their sizes, from largest to smallest • Several functions discovered: – Interaction with the core promoter elements – Interaction with gene-specific transcription factors – When attached to TBP extend the binding of TFIID beyond the TATA box 11-10
Model for the Interaction Between TBP and Promoters 11-11
Roles of TAF1 and TAF2 • The TAF1 and TAF2 help the TFIID bind to the initiator and DPE of promoters • They enable TBP to bind to TATA-less promoters that contain elements such as a GC box • Different combinations of TAFs are required to respond to variosu activators, at least in higher eukaryotes • TAF1 has two enzymatic activities: – Histone acetyltransferase (HAT) – Protein kinase 11-12
Transcription Enhancement by Activators 11-13
Exceptions to the Universality of TAFs and TBP • TAFs are not universally required for transcription of class II genes • Even TBP is not universally required • Some promoters in higher eukaryotes respond to an alternative protein such as TRF1 (TBP-related factor 1) • The general transcription factor NC2: – Stimulates transcription from DPE-containing promoters – Represses transcription from TATA-containing promoters 11-14
Structure and Function of TFIIB • Structural studies have revealed that TFIIB binds to TBP at the TATA box via its C- terminal domain and polymerase II via its N-terminal domain • The protein provides a bridging action that effects a coarse positioning of polymerase active center about 25 –30 bp downstream of the TATA box • Plays an important role in establishing the transcription start site 11-15
TFIIB Domains • A loop motif of the N-terminal domain in TFIIB effects a fine positioning of the transcription start by interacting with template ssDNA near the active center • TFIIB N-terminal domain, finger and linker domains, lies close to the RNA polymerase II active center and to largest subunit of TFIIF in preinitiation complex 11-16
TFIIH • TFIIH is the last general transcription factor to join the preinitiation complex (contains 9 subunits) • Separates into 2 complexes • Protein kinase complex of 4 subunits • Core TFIIH complex of 5 subunits with 2 DNA helicase/ATPase activities • Plays two major roles in transcription initiation: – Phosphorylates the CTD of RNA polymerase II – Unwinds DNA at the transcription start site to create the “transcription bubble” 11-17
Phosphorylation of the CTD of RNA Polymerase II • The preinitiation complex forms with hypophosphorylated form of RNA polymerase II (IIA) • Then TFIIH phosphorylates serines 2 and 5 in the heptad repeat in the carboxyl- terminal domain (CTD) of the largest RNA polymerase subunit – This creates the phosphorylated form of the polymerase enzyme (IIO) – This phosphorylation is essential for initiation of transcription 11-18
Phosphorylated Polymerase IIO During Elongation • During the shift from initiation to elongation, two serines of the CTD are phosphorylated (serines 2 and 5 - and sometimes serine 7) • Evidence exists that transcription complexes near the promoter have CTDs in which serine 5 is phosphorylated but that this phosphorylation shifts to serine 2 as transcription progresses • TFIIH phosphorylates serine 5 and CTDK-1 (in yeast) phosphorylates serine 2 11-19
Role of TFIIE and TFIIH TFIIE and TFIIH are not essential for the formation of an open promoter complex or for elongation • Required for promoter clearance • TFIIH has DNA helicase activity that is essential for transcription, presumably because it causes full melting of the DNA at the promoter and thereby facilitates promoter clearance 11-20