Lecture 2: RNA polymerase Flashcards
What is RNA polymerase?
- The enzyme can be found as the core RNA polymerase.
- It can also be found as a holoenzyme. The holoenzyme is the cofactor and enzyme together. In this case it is subunit σ which is the cofactor.
- The rest of the enzyme consists of 2 α subunits, 1 β subunit, 1 β’ subunit and 1 ω subunit.
- The holoenzyme is required for initiating transcription. The enzyme has a higher affinity for the start site than DNA in general.
- The core enzyme is involved elongation. The enzyme has to move away from the start site after initiation, so it requires lower affinity.
- The σ subunit is involved in initial binding but not elongation.
What are σ subunits?
The σ subunit finds a promoter and melts the DNA.
• σ factors bind at the -35 upstream recognition sequence and at the -10 downstream recognition sequence.
• There is also a 17bp spacer required for proper bending of DNA over the polymerase (for σ70).
• There are 2 general classes of σ factors: σ70 and σ54.
• σ70 is involved in housekeeping genes at exponential growth.
• σ54 is involved in nitrogen-regulated genes.
How is transcription initiated? Draw the rate diagram of how this happens.
Initiation involves bind to the promoter using σ subunits.
1) The enzyme (E) binds at a random point in the DNA. It is transferred to the promoter site by sliding along the DNA by transfer from one DNA loop to another.
2) The enzyme makes contacts with key promoter elements to form the closed complex (EPc). Binding of the enzyme to DNA can be described by the binding constant K¬B¬ (this is normally around 107 to 109 M-1.
3) The DNA at the promoter is melted to facilitate initiation. This melting is described by the kinetic constant k+2 (this is usually 10-1 to 10-3 s-1).
4) The holoenzyme forms at least 4 successive complexes with DNA before the polymerase initiates transcription. Evidence for these comes from studies of the ability of the enzyme to protect DNA from attack from agents like hydroxyl radicals, methylating agents and DNase1.
What is σ70? Draw a diagram of sigma subunits.
σ70 is involved in housekeeping genes.
• σ70 has 4 distinct regions.
• σ2/σ4 regions bind to the -10 and -35 sequences. These are helix turn helix motifs.
• Initially σ is a double strand DNA binding protein.
• Other regions of the polymerase (α subunits, σ1 and σ3) may contact the promoter.
• The σ3.2 loop protrudes into the RNAP active site channel and out underneath the β flap. It stabilises the binding of the initiating nucleotide substrate.
• It is not required after the first phosphodiester bond is formed, when the corresponding site is occupied by the 3’ end of the growing RNA product.
• This region also promotes abortive initiation by blocking the path of the nascent RNA into the exit channel.
• σ1.1 is positioned in the active-site channel through electrostatic interactions.
• Protection studies show that RPc protects DNA from -55 to -6.
How do the closed and open complexes form?
- The closed complex has the enzyme binding to double stranded DNA. The open complex binds to single stranded DNA.
- Protects a region from -55 to +20. Much of the DNA is now inside the enzyme.
- The open complex is resistant to heparin displacing the DNA. However, it is sensitive to inhibition by rifampicin.
- The conformational change allows interactions of ss binding sites with DNA.
- This leads to a gain in binding energy. This interaction involves aromatic residues in σ.
- The σ factor catalyses the formation of a single stranded promoter state which is required for RNA synthesis.
- We can distinguish closed and open using heparin and rifampicin respectively.
What are anti-σ factors (ASFs)?
- ASFs are proteins that bind to σ factors and inhibit transcriptional activity.
- They are often transmembrane proteins that bind to and inhibit their cognate σ factors.
- Upon receiving a stimulus, the σ factor is released. It binds to RNAP to stimulate transcription from the specific subset of genes.
- AsiA is an ASF for σ70 in E. coli that binds to σ4.2 and blocks binding to -35. Promoters lacking -35 regions are less sensitive to AsiA inhibition. AsiA is produced by T4 bacteriophages so it can control gene transcription.
What do the rate constants tell us?
- Each promoter has an inherent K¬B (association constant of RNAP binding and a k+2 (rate constant for opening of the duplex DNA in the promoter/polymerase complex).
- These two constants determine promoter strength (frequency of initiation of transcription in the absence of accessory factors).
- They are not in themselves regulatory features.
- They ensure the different genes are expressed to very different extents depending on the needs of the cell.
- For example, a cell will always if growing need to make a lot rRNA and a small amount of lac repressor.
