The Mechanism of Transcription in Bacteria

Chapter 6

RNA Polymerase Structure• 1969 SDS-PAGE of E.coli RNA polymerase

- 2 very large subunits are β (150 kD) and β’ (160 kD)

– Sigma (σ) at 70 kD

– Alpha (α) at 40 kD – 2 copies present in holoenzyme

– Omega (ω) at 10 kD

Specificity factor

• Holoenzyme and Core enzyme

• Core enzyme transcribes both DNA strands

• Without σ-subunit the core enzyme has basic transcribing ability but lacks specificity

Promoters

• Presence of the σ-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 σ-subunit

Binding of RNA Polymerase to Promoters

• Experiment measures binding of DNA to enzyme using nitrocellulose filters

– Holoenzyme binds filters tightly

– Core enzyme binding is more transient

Temperature and RNA Polymerase Binding

• As temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically

• Higher temperature promotes DNA melting

Promoter Sequence

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

Promoter Structure

• 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

rrnB promoter in E.coli

• UP promoter element stimulating transcription by a factor of 30

• UP is associated with 3 “Fis” sites.

Polymerase/Promoter Binding

• Holoenzyme binds DNA loosely

• Complex loosely bound at promoter = closed promoter complex - dsDNA in closed form

• Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound

Initiation and Elongation Transcription

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

Functions of σ

• Gene selection for transcription by σ causes tight binding between RNA polymerase and promoters

• Tight binding depends on local melting of DNA that permits open promoter complex

• Dissociation of σ from core after sponsoring polymerase-promoter binding

Reuse of σ

• During initiation σ can be recycled for additional use in a process called the σ cycle

• Core enzyme can release σ which then associates with another core enzyme

σ May Not Dissociate from Core During Elongation

• The σ -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

Local DNA Melting at the Promoter

• 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

• Size of the DNA transcription bubble in complexes where transcription was active –

17-18 bp

Structure and Function of σ

• Genes encoding a variety of σ -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

Homologous Regions in Bacterial σ Factors

Region 1

• Role of region 1 appears to be in preventing σ from binding to DNA by itself

• This is important as σ binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription

Region 2

• Most highly conserved of the four

• Four subregions – 2.1 to 2.4

• 2.4 recognizes the promoter’s -10 box

• The 2.4 region appears to be α-helix

Region 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 DNA-

binding domain and appears to govern binding to the -35 box of the promoter

Role of α-Subunit in UP Element Recognition

• RNA polymerase binds to a core promoter via its σ-factor - no help from C-terminal domain of α-subunit

• Binds to a promoter with an UP element using σ plus the α -subunit C-terminal domains

• Results in very strong interaction between polymerase and promoter

• This produces a high level of transcription

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

Function of the Core Polymerase

• Core polymerase contains the RNA synthesizing machinery

• Phosphodiester bond formation involves the β- and β’-subunits

• β- and β’-subunits also participate in DNA binding

• Assembly of the core polymerase is a major role of the α-subunit

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

Termination of transcription

• 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 - ρ -dependent terminators

Chain Termination• Two types of termination mechanisms:

• intrinsic termination- controlled by specific sequences, termination sites

• Termination sites characterized by two inverted repeats

Rho-Independent Termination

• The repeat at right is symmetrical around its center shown with a dot

• A transcript of this sequence is self-complementary– Bases can pair up to

form a hairpin

– Inverted Repeats and Hairpins

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

Mechanism of ρ dependent termination

• Binding to the growing transcript - ρ follows the RNA polymerase

• Catches the polymerase as it pauses at the hairpin

• Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid

• This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis:

• against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and

• against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

Disclaimer• This workforce solution was funded by a grant awarded under the

President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.