Bacterial Transcription

Table of Contents

The transcription reaction is divided into three stages: Initiation, Elongation, and Termination.

Transcription begins with RNA polymerase binding to the promoter sequence on the DNA double helix.
The RNA polymerase moves along the template strand, extending the growing RNA chain in the 5′-to-3′ direction by the stepwise addition of ribonucleoside triphosphates until it reaches a termination signal, at the point the newly synthesized RNA chain and the polymerase are released from the DNA.

Initiation is the process of synthesizing the first nucleotide bonds in RNA.
The promoter sequence of DNA defines the sequence needed for RNA polymerase to bind to the template and initiate the reaction.
A gene promoter is a cis-acting, position-dependent DNA sequence necessary for initiating transcription of the gene.

The DNA sequence of the promoter region is recognized by the requisite RNA polymerase.
The best characterized bacterial promoters are those of E.coli recognized by σ⁷⁰, containing two 6-base pairs consensus sequences (-10 and -35 sequences), where the -10 sequence has the consensus TATAAT and the -35 sequence has consensus TTGACA.
The -10 and -35 regions are designated to reflect their approximate distances in nucleotides from the start site, with the distance between them being 16 to 18 bp and the -10 hexamer lying 7 bp upstream of the start point.

Strong promoters have an AT-rich sequence located in the upstream region, called the UP element, that interacts with the α subunit of RNA polymerase.
Most E.coli promoters interact with the major form of RNA polymerase, containing σ⁷⁰.
Transcription of certain groups of genes is carried out by E.coli RNA polymerases containing one of several alternative sigma factors (-σ²⁸, σ³², σ³⁸, and σ⁵⁴), recognizing different consensus promoter sequences than σ⁷⁰.

The holoenzyme promoter reaction starts by forming a closed binary complex where the DNA remains duplex.
RNA polymerase holoenzyme initially binds to DNA, covering some 75-80 bp, extending from -55 to +20.
The closed complex is converted into an open complex by melting a short region of DNA within the sequence bound by the enzyme.

The length of the transcription bubble created by a local unwinding is -12-14 bp.
The first two nucleotides are incorporated, and a phosphodiester bond forms between them, generating a ternary complex containing RNA as well as DNA and enzyme.
Only the holoenzyme can initiate transcription.

Core enzyme can synthesize RNA on a DNA template, but cannot initiate transcription at the proper sites.
RNA synthesis can start de novo, without the requirement for a primer.
The first nucleotide present in the transcript at the 5′ end is either pppG or pppA.

RNA synthesis is frequently aborted after usually 2 or 3 but up to 10 to 12 nucleotides have been joined, a phenomenon is known as abortive initiation.
Once a polymerase manages to make an RNA longer than 10 bp, a stable ternary complex is formed containing the enzyme, the DNA template, and a growing RNA chain.

Transcription initiation in bacteria such as E.coli is controlled in two distinct ways: constitutive control, which depends on the structure of the promoter, and regulatory control, which depends on the influence of regulatory proteins.

Promoter structure determines the basal level of transcription initiation.
The consensus sequence for the E.coli promoter is quite variable.
Different promoters vary in their efficiency, defined as the number of productive initiations that are promoted per second, where a productive initiation results in the RNA polymerase clearing the promoter and beginning the synthesis of a full-length transcript.

based on the nature of consensus sequences, the promoter may be strong or weak.
Strong promoters support high rates of transcription initiation, and most of the promoters in E.coli are strong promoters.

Enzyme moves along DNA and extends the growing RNA chain.
Unwinds DNA helix to expose a new segment of template in single-stranded condition.
Nucleotides covalently added to the 3′ end of the growing RNA chain, forming an RNA-DNA hybrid.

Length of RNA-DNA hybrid within the open complex is ~8-9 bp.
During each nucleotide addition, β- and γ-phosphates are removed from the incoming nucleotide, and the hydroxyl group is removed from the 3′-carbon of the nucleotide at end of chain.
Overall reaction rate is ~40 nucleotides/second at 37°C.

Unwinding and rewinding occur as RNA polymerase transcribes DNA.
Positive supercoils generated ahead, negative supercoils left behind.
Gyrase introduces negative supercoils, and topoisomerase I remove negative supercoils.

Cordycepin, an adenosine analog lacking the 3′-OH group, inhibits elongation by preventing further RNA chain elongation.

Recognition of the point at which no further bases should be added to the chain.
Sequence of DNA required for these reactions is called the terminator.

Enzyme stops adding nucleotides to growing RNA chain, releases completed product and dissociates from DNA template.
Bacteria use two distinct strategies for transcription termination: intrinsic and Rho-dependent.
Intrinsic terminators promote the dissociation of polymerase by destabilizing the attachment of the growing transcript to the template.

Transcript forms hairpin structure by forming complementary base pairing.
Intrinsic terminators consist of G-C-rich hairpin in RNA product followed by U-rich region in which termination occurs.
Typical distance between the hairpin and U-rich region is 7-9 bases.

Rho-dependent termination requires the activity of a protein called Rho.
Rho is an ATP-dependent RNA-stimulated helicase that disrupts nascent RNA-DNA complex
Rho is a ~275 kDa hexamer of identical subunits.

Rho binds to RNA and translocates along RNA until it reaches the RNA-DNA hybrid in RNA polymerase, where it releases RNA from DNA.

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