Lecture Molecular biology (Fifth Edition): Chapter 6 - Robert F. Weaver

Chia sẻ: Nnmm Nnmm | Ngày: | Loại File: PPT | Số trang:55

Thêm vào BST

Báo xấu

77
lượt xem 3
download

Download Vui lòng tải xuống để xem tài liệu đầy đủ

Chapter 6 - The mechanism of transcription in bacteria. In this chapter we will focus on the basic mechanism of transcription. We will begin with RNA polymerase, the enzyme that catalyzes transcription. We will also look at the interaction between RNA polymerase and DNA.

Chủ đề:

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

Lưu

Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 6 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 6 The Mechanism of Transcription in Bacteria Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits – 2 very large subunits are (150 kD) and ’ (160 kD) – Sigma ( ) at 70 kD – Alpha ( ) at 40 kD – 2 copies present in holoenzyme – Omega (w) at 10 kD • Was not clearly visible in SDS-PAGE, but seen in other experiments • Not required for cell viability or in vivo enzyme activity • Appears to play a role in enzyme assembly 6-2
Sigma as a Specificity Factor • Core enzyme without the subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA • With subunit, the holoenzyme worked equally well on both types of DNA 6-3
Summary • The key player in the transcription process is RNA polymerase • The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a -factor, which directs the core to transcribe specific genes 6-4
6.2 Promoters • Why was the core RNA polymerase capable of transcribing nicked DNA in the previous table? • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • The presence of the -subunit permits recognition of authentic RNA polymerase binding sites called promoters • Transcription that begins at promoters is specific, directed by the -subunit 6-5
Binding of RNA Polymerase to Promoters • How tightly does core enzyme v. holoenzyme bind DNA? • Experiment measures binding of DNA to enzyme using nitrocellulose filters – Holoenzyme binds filters tightly – Core enzyme binding is more transient 6-6
Temperature and RNA Polymerase Binding • As the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperatures promote DNA melting and encourage RNA polymerase binding 6-7
RNA Polymerase Binding Hinkle and Chamberlin proposed: • RNA polymerase holoenzyme binds DNA loosely at first – Binds at promoter initially – Scans along the DNA until it finds a promoter • Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form • Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA 6-8
Polymerase/Promoter Binding • Holoenzyme binds DNA loosely at first • Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form • Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound 6-9
Summary • The -factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter • This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by • The -factor can therefore select which genes will be transcribed 6-10
Core Promoter Elements • There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box • Another short sequence centered 35 bp upstream is known as the -35 box • Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes 6-11
Promoter Strength • Consensus sequences: – -10 box sequence approximates TATAAT – -35 box sequence approximates TTGACA • Mutations that weaken promoter binding: – Down mutations – Increase deviation from the consensus sequence • Mutations that strengthen promoter binding: – Up mutations – Decrease deviation from the consensus sequence 6-12
UP Element • The UP element is upstream of the core promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for the transcription- activator protein Fis, not for the polymerase itself 6-13
The rrnB P1 Promoter • Transcription from the rrn promoters respond –Positively to increased concentration of iNTP –Negatively to the alarmone ppGpp 6-14
6.3 Transcription Initiation • Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond • Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA • Abortive transcripts such as these have been found up to 10 nt 6-15
Stages of Transcription Initiation • Formation of a closed promoter complex • Conversion of the closed promoter complex to an open promoter complex • Polymerizing the early nucleotides – polymerase at the promoter • Promoter clearance – transcript becomes long enough to form a stable hybrid with template 6-16
Sigma Stimulates Initiation of Transcription • In this first experiment stimulation by appears to cause both initiation and elongation • Or stimulating initiation by provides more initiated chains for core polymerase to elongate • Further experiments by the same group proved that does not stimulate elongation 6-17
Reuse of • During initiation can be recycled for additional use with a new core polymerase • The core enzyme can release which is then free to associate with another core enzyme 6-18
Fluorescence Resonance Energy Transfer • The -factor changes its relationship to the core polymerase during elongation • It may not dissociate from the core but actually shift position and become more loosely bound to core • To answer this question Fluorescence Resonance Energy Transfer (FRET) was used as it relies on two fluorescent molecules that are close enough together to engage in transfer of resonance energy • FRET allows the position of relative to a site on the DNA to be measured without using separation techniques that might displace from the core enzyme 6-19
FRET Assay for Movement Relative to DNA 6-20