RNA polymerase

Lecture8 RNA Polymerase Structure & Function 1 •Molecular Genetics I: MLGN 301 2016 23/11/2016 By Prof.Dr /Ahmed Mansour Alzohairy

Genetics Department, Zagazig University, Zagazig, Egypt

Recommended book for further information

Lecture PowerPoint to accompany

Molecular Biology Fourth Edition

Robert F. Weaver Chapter 3 An Introduction to Gene Function Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Learning objectives

Learning about: • RNA Polymerase Structure • Promoters • Transcription Initiation

Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

Molecular Genetics I: MLGN 301 2016 Trainer name: Prof. Ahmed Mansour Alzohairy

Learning outcomes By the end of this session and practical, students are expected to be able to understand what are: •Sigma as a Specificity Factor •Binding of RNA Polymerase to Promoters •Temperature and RNA Polymerase Binding •RNA Polymerase Binding •Core Promoter Elements •Promoter Strength •Stages of Transcription Initiation •Sigma Stimulates Transcription Initiation •Sigma May Not Dissociate from Core During Elongation


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6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits – 2 very large subunits are b (150 kD) and b’ (160 kD) – Sigma (s) at 70 kD – Alpha (a) 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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Sigma as a Specificity Factor

• Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA • With s subunit, the holoenzyme worked equally well on both types of DNA


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Testing Transcription • Core enzyme transcribes both DNA strands • Without s-subunit the core enzyme has basic transcribing ability but lacks specificity


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6.2 Promoters • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • Presence of the s-subunit permitted recognition of authentic RNA polymerase binding sites • Polymerase binding sites are called promoters • Transcription that begins at promoters is specific, directed by the s-subunit Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Temperature and RNA Polymerase Binding • As temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperature promotes DNA melting


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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 one

• 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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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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 for each of these boxes


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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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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UP Element • UP element is a promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for transcription-activator protein Fis, not for the polymerase itself • Transcription from the rrn promoters respond – Positively to increased concentration of iNTP – Negatively to the alarmone ppGpp Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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The rrnB P1 Promoter


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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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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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 Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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The Functions of s • Gene selection for transcription by s causes tight binding between RNA polymerase and promoters • Tight binding depends on local melting of DNA that permits open promoter complex • Dissociation of s from core after sponsoring polymerase-promoter binding Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Sigma Stimulates Transcription Initiation • Stimulation by s appears to cause both initiation and elongation • Or stimulating initiation provides more initiated chains for core polymerase to elongate Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Reuse of s

• During initiation s can be recycled for additional use in a process called the s cycle • Core enzyme can release s which then associates with another core enzyme Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Sigma May Not Dissociate from Core During Elongation • The s-factor changes its relationship to the core polymerase during elongation • It may not dissociate from the core • May actually shift position and become more loosely bound to core


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Fluorescence Resonance Energy Transfer

• Fluorescence resonance energy transfer (FRET) relies on the fact that two fluorescent molecules close together will engage in transfer of resonance energy • FRET allows the position of s relative to a site on the DNA to be measured with using separation techniques that might displace s from the core enzyme Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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FRET Assay for s Movement Relative to DNA


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Local DNA Melting at the Promoter • From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp • In another experiment, the length of the melted region was found to be 12 bp • Later, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Region of Early Promoter Melted by RNA Polymerase


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Structure and Function of s • Genes encoding a variety of s-factors have been cloned and sequenced • There are striking similarities in amino acid sequence clustered in 4 regions • Conservation of sequence in these regions suggests important function • All of the 4 sequences are involved in binding to core and DNA Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Homologous Regions in Bacterial s Factors


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E. coli s70 • Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements, -10 box and –35 box are notes Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Region 1 • Role of region 1 appears to be in preventing s from binding to DNA by itself • This is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Region 2 • This region is the most highly conserved of the four • There are four subregions – 2.1 to 2.4 • 2.4 recognizes the promoter’s -10 box • The 2.4 region appears to be a-helix


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Regions 3 and 4 • Region 3 is involved in both core and DNA binding • Region 4 is divided into 2 subregions – This region seems to have a key role in promoter recognition – Subregion 4.2 contains a helix-turn-helix DNAbinding domain and appears to govern binding to the -35 box of the promoter Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Summary • Comparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sources • Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition • The s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA-binding region of s • Region between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Practical (to try in your own time)

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What are the identified RNA polymerase subunits from E. coli ?

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What is the main function of RNA polymerase sigma subunit?

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What is effect of Temperature on RNA Polymerase Binding?

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What is the differences between closed promoter complex & Open promoter complex?

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Describe with drawing two Core Promoter Elements and UP element?

- What are the differences in Structure and Function between sigma subunit 4 regions? Molecular Genetics I: MLGN 301 2016 Trainer name: Prof. Ahmed Mansour Alzohairy





Department of Genetics, Zagazig University, Zagazig, Egypt