RNA polymerase

Lecture9 RNA Polymerase Structure & Function 2 •Molecular Genetics I: MLGN 301 2016 30/11/2016 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:

• More about a-Subunit • Elongation • Termination of Transcription 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: •Role of a-Subunit in UP Element Recognition •Modeling the Function of the C-Terminal Domain •Function of the Core Polymerase •Role of b in Phosphodiester Bond Formation •Role of b’ and b in DNA Binding •Strategy to Identify Template Requirements •Observations Relating to Polymerase Binding •Structure of the Elongation Complex •RNA-DNA Hybrid •Structure of the Core Polymerase and Structure of the Holoenzyme •Rho-Independent Termination Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Role of a-Subunit in UP Element Recognition • RNA polymerase itself can recognize an upstream promoter element, UP element • While s-factor recognizes the core promoter elements, what recognizes the UP element? • It appears to be the a-subunit of the core polymerase Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Modeling the Function of the CTerminal Domain

• RNA polymerase binds to a core promoter via its s-factor, no help from C-terminal domain of asubunit • Binds to a promoter with an UP element using s plus the a-subunit C-terminal domains • Results in very strong interaction between polymerase and promoter • This produces a high level of transcription

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

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6.4 Elongation • After transcription initiation is accomplished, core polymerase continues to elongate the RNA • Nucleotides are added sequentially, one after another in the process of elongation


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Function of the Core Polymerase • Core polymerase contains the RNA synthesizing machinery • Phosphodiester bond formation involves the band b’-subunits • These subunits also participate in DNA binding • Assembly of the core polymerase is a major role of the a-subunit Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Role of b in Phosphodiester Bond Formation

• Core subunit b lies near the active site of the RNA polymerase • This active site is where the phosphodiester bonds are formed linking the nucleotides • The s-factor may also be near nucleotidebinding site during initiation phase Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Role of b’ and b in DNA Binding • In 1996, Evgeny Nudler and colleagues showed that both the b- and b’-subunits are involved in DNA binding • They also showed that 2 DNA binding sites are present – A relatively weak upstream site • DNA melting occurs • Electrostatic forces are predominant

– Strong, downstream binding site where hydrophobic forces bind DNA and protein together


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Strategy to Identify Template Requirements


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Observations Relating to Polymerase Binding • Template transfer experiments have delineated two DNA sites that interact with polymerase • One site is weak – It involves the melted DNA zone, along with catalytic site on or near b-subunit of polymerase – Protein-DNA interactions here are mostly electrostatic and are salt-sensitive

• Other is strong binding site involving DNA downstream of the active site and the enzyme’s b’- and b-subunits Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Structure of the Elongation Complex • How do structural studies compare with functional studies of the core polymerase subunits? • How does the polymerase deal with problems of unwinding and rewinding templates? • How does it move along the helical template without twisting RNA product around the template? Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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RNA-DNA Hybrid • The area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNA • In T7 the similar hybrid appears to be 8 bp long


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Structure of the Core Polymerase • X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw • It appears designed to grasp the DNA • A channel through the enzyme includes the catalytic center – Mg2+ ion coordinated by 3 Asp residues – Rifampicin-binding site Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Structure of the Holoenzyme • Crystal structure of T. aquaticus RNA polymerase holoenzyme shows an extensive interface between s and b- and b’-subunits of the core • Structure also predicts s region 1.1 helps open the main channel of the enzyme to admit dsDNA template to form the closed promoter complex • After helping to open channel, the s will be expelled from the main channel as the channel narrows around the melted DNA of the open promoter complex Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Additional Holoenzyme Features • Linker joining s regions 3 and 4 lies in the RNA exit channel • As transcripts grow, they experience strong competition from s3-s4 linker for occupancy of the exit channel


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Structure of the Holoenzyme-DNA Complex Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals: – DNA is bound mainly to s-subunit – Interactions between amino acids in region 2.4 of s and 10 box of promoter are possible – 3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted – 2 invariant basic amino acids in s predicted to function in DNA binding are positioned to do so – A form of the polymerase that has 2 Mg2+ ions Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Topology of Elongation • Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA • Polymerase maintains a short melted region of template DNA • DNA must unwind ahead of the advancing polymerase and close up behind it • Strain introduced into the template DNA is relaxed by topoisomerases Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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6.5 Termination of Transcription • When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA • There are 2 main types of terminators – Intrinsic terminators function with the RNA polymerase by itself without help from other proteins – Other type depends on auxiliary factor called r, these are r-dependent terminators Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Rho-Independent Termination • Intrinsic or r-independent termination depends on terminators of 2 elements: – Inverted repeat followed immediately by – T-rich region in nontemplate strand of the gene

• An inverted repeat predisposes a transcript to form a hairpin structure


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Inverted Repeats and Hairpins • The repeat at right is symmetrical around its center shown with a dot • A transcript of this sequence is selfcomplementary – Bases can pair up to form a hairpin as seen in the lower panel Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Structure of an Intrinsic Terminator

• Attenuator contains a DNA sequence that causes premature termination of transcription • The E. coli trp attenuator was used to show:

– Inverted repeat allows a hairpin to form at transcript end – String of T’s in nontemplate strand result in weak rU-dA base pairs holding the transcript to the template strand


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Model of Intrinsic Termination Bacterial terminators act by: • Base-pairing of something to the transcript to destabilize RNA-DNA hybrid – Causes hairpin to form

• Causing the transcription to pause – Causes a string of U’s to be incorporated just downstream of hairpin Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Rho-Dependent Termination • Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro • This depression was due to termination of transcription • After termination, polymerase must reinitiate to begin transcribing again


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Rho Affects Chain Elongation • There is little effect of r on transcription initiation, if anything it is increased • The effect of r on total RNA synthesis is a significant decrease • This is consistent with action of r to terminate transcription forcing time-consuming reinitiation Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Rho Causes Production of Shorter Transcripts

• Synthesis of much smaller RNAs occurs in the presence of r compared to those made in the absence • To ensure that this due to r, not to RNase activity of r, RNA was transcribed without r and then incubated in the presence of r • There was no loss of transcript size, so no RNase activity in r Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Rho Releases Transcripts from the DNA Template

• Compare the sedimentation of transcripts made in presence and absence of r

– Without r, transcripts cosedimented with the DNA template – they hadn’t been released – With r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template

• It appears that r serves to release the RNA transcripts from the DNA template Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Mechanism of Rho No string of T’s in the r- • dependent terminator, just inverted repeat to hairpin Binding to the growing • transcript, r follows the RNA polymerase It catches the polymerase • as it pauses at the hairpin Releases transcript from • the DNA-polymerase complex by unwinding the RNA-DNA hybrid Robert F. Weaver. Molecular Biology Fourth Edition. Copyright © The McGraw-Hill Companies, Inc.

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Practical (to try in your own time)

• Describe the different Roles of RNA polymerase: – Alpha (a)

– Beta b and b’ – Sigma (s) – Rho (r) • Describe the differences between the 2 main types of terminators of RNA polymerization?






Department of Genetics, Zagazig University, Zagazig, Egypt