IT3: Transcription Flashcards
How may stochastic gene transcription arise in eukaryotic cells?
A major source of variation in eukaryotes is chromatin which can control access of TFs to their binding sites.
State the 3 methods used in bacteria to transduce a signal.
Anti-sigma factors
2-component regulatory systems
Individual activators and repressors
How does bacterial RNAP differ from eukaryotic RNAP?
Bacteria:
- Consists of a core enzyme and a sigma factor that makes up the holoenzyme
- Can initiate transcription on its own
- Primarily regulated by sigma factor availability
Eukaryotic:
- Consists of several subunits that form the core enzyme, along with accessory factors
- Requires additional factors (e.g., GTFs and Mediator) to initiation transcription
- Regulated by TFs, chromatin modifiers, and signaling pathways
Compare sigma70 and sigma54
Sigma70 recognizes promoters at -10 (Pribnow box) and -35. It can initiate transcription without assistance (except at the lac operon).
Sigma54 is involved in nitrogen-regulated genes, binding at -12 and -24 and requires bacterial enhancer binding proteins to ‘melt’ the DNA, using ATP. In this way, bEPB acts like eukaryotic TFIIH as both require ATP.
What are the roles of the 3 types of eukaryotic RNAPs?
Pol I transcribes rRNA genes.
Pol II transcribes mRNA.
Pol III transcribes tRNAs and snRNAs.
What is the pre-initiation complex?
The preinitiation complex is a complex of approximately 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea. The preinitiation complex positions RNA polymerase II at gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription.
The minimal PIC includes RNA polymerase II and six general transcription factors. Additional regulatory complexes (such as the mediator coactivator and chromatin remodeling complexes) may also be components of the PIC.
How does RNAP find and bind to a promoter in prokaryotes?
- RNAP binds on and off to random DNA sequences, scanning until it finds promoter sequences.
- -10 and -35 need to be contacted at the same time, resulting in distortion of the DNA.
- Introducing this torsional stress forces the DNA to melt to relieve the strain, temporarily becoming single-stranded. This is the open complex.
Describe the steps of how sigma70 leads to elongation.
- Sigma70 is required to melt the DNA
- Loop 3.2 of sigma70 is required for the formation of the first phosphodiester bond
- It promotes abortive initiation but prevents the move to the elongating form of RNAP.
- Must be displaced from RNAP to elongate.
What is the role of the sigma70 3.2 loop?
Protrudes into the RNAP active site, stabilizing the binding of the initiating nucleotide substrate and promoting abortive initiation by blocking the path of the nascent RNA into the exit channel.
Describe abortive initiation
Abortive initiation is an obligatory step in both bacteria and eukaryotes that helps RNAP to ensure it has properly positioned itself at the TSS.
RNAP synthesizes ~9 nucleotides in its holoenzyme form, but due to the sigma70 3.2 loop blocking the exit channel, the DNA collects within a pocket of the enzyme. This causes distortion and increases the strain of the interaction between the DNA and the enzyme. Relaxation can be achieved by releasing RNA, thus aborting transcription.
If everything is correct, the sigma factor is released and RNAP enters elongation.
How does sigma70 melt the DNA at the lac operon?
The lac operon has a non-consensus sequence - instead of the Pribnow box being AT rich, it contains a GC link that prevents sigma70 from binding.
Activators Crp/CAP improve the Kb by providing additional contacts for RNAP, and k+2 by further distorting/bending the DNA.
Describe the mechanism of elongation in transcription.
Spt5 (NusG in bacteria) is an elongation factor that binds the polymerase clamp to help stabilize the interaction between RNAP and the DNA, enhancing processivity and preventing premature termination. It can only associate upon promoter escape because its binding site is occupied in the initiation complex.
During the NAC, an NTP substrate binds to the open active center before occupying the insertion site as the trigger loop folds to close the active center. This leads to catalytic nucleotide incorporation. Release of PPi may cause trigger loop unfolding and opening of the active site. The bridge helix works with the trigger loop to shift the newly integrated NTP out of the active site.
How does the trigger loop contribute to accuracy?
It eases the barriers to backtracking - a mechanism for removing mis-incorporated nucleotides.
What is the function of the RNAP clamp?
It is initially in a more open position, but after DNA has been melted, it gets repositioned to keep the DNA in the active site tightly (i.e., it ‘clamps’ down onto the DNA).
What is the elemental pause state of RNAP?
A step during elongation where the enzyme undergoes brief stalling. This is a highly conserved process that allows for the recruitment of various elongation factors and regulatory proteins to modulate RNAP activity e.g., NusA and Spt5.
Pausing arises when nucleic acid interactions in the elongation complex lead to a loosening of the clamp domain and incomplete translocation, trapping RNAP in the paused state. The hairpin-stabilized pause is generated from an elemental pause when the nascent RNA folds into a hairpin that jams open the clamp domain and traps the trigger loop in an inactive conformation.
Describe Rho-independent termination.
(Intrinsic termination) occurs in bacteria using a terminator motif that’s present in the DNA template. This motif usually contains an inverted repeat followed by a U-rich tract, resulting in the formation of a stem-lop in the RNA transcript. This causes pausing of RNAP and destabilization of the RNAP:DNA:RNA complex.
Destabilization triggers release of mRNA, leading to termination.
RNA that’s tightly associated with ribosomes can’t form the pausing stem-loop structures for termination.
Describe Rho-dependent termination.
Rho is an ATPase that travels with some elongating RNAPs and pulls the RNA out of RNAP, terminating transcription by extraction.
How can cysteine levels alter transcription of the ubiGmccBA operon?
The 5’UTR is able to form either a terminator or anti-terminator structure depending on the cys-tRNA presence. The presence of cys-tRNA signals that there are high levels of cysteine, and so the terminating structure is formed.
If the cell is low in cys-tRNA, but high in SAM, the uncharged tRNA prevents terminator loop formation, and the structure is altered to allow termination of the antisense strand, giving high expression of structural genes to convert SAM into cysteine.
If there’s low cys-tRNA levels and low SAM levels, the uncharged tRNA binds to the 5’UTR and prevents terminator structure formation. This allows for synthesis of the operon that encodes enzymes needed to synthesize cysteine from methionine.
How does attenuation differ from antitermination?
Attenuation:
- Terminates transcription
- Occurs when transcription is slower than translation, allowing the ribosome to catch up to RNAP, causing formation of an RNA secondary structure that prematurely terminates transcription
- Can be mediated by ribosomes, proteins, or uncharged tRNA
In trp operon, NusG and NusA aid termination by stabilizing the hairpin and stopping translocation.
Antitermination:
- Allows transcription to continue beyond a termination site
- Can occur in response to environmental or cellular signals, mediated by factors such as NusA and Spt5
- Factors prevent formation of termination structure
Why may RNAP pause and what can this lead to?
- Rewinding of the DNA supplies little energy (A-T rich) and unwinding of the downstream DNA requires more energy (G-C rich), reducing rate of elongation and pausing.
- Weak base-pairing in the hybrid region compared to upstream e.g., A-U.
In these circumstances, the DNA:RNA hybrid may return upstream whilst the downstream edge rewinds, leading to backtracking.
This causes displacement of the 3’ end from the active site and so this end must be cleaved to generate a new 3’OH for synthesis to continue.
How does a promoter differ from an enhancer?
Promoter:
- Binding site for RNAP and other TFs.
- Located near the TSS
- H3K4me3 and H3K27ac
Enhancer:
- Bind TFs to enhance transcription
- Located upstream or downstream of TSS
- Can confer tissue- or developmental-specific expression patterns.
- H3K4me1 and H3K27ac
What is a proto-enhancer?
Modular components of an enhancer that each bind a TF. Each proto-enhancer is made up of an enhanson - the fundamental units of enhancer function. e.g., the SV40 enhancer.
What is a super enhancer/LCR?
A large cluster of enhancers that control the expression of genes that are critical for cell identity and differentiation. The best characterized of these are the beta-globin loci.
What are co-activators and co-repressors? What 2 models describe how they might work?
Co-activators such as Mediator facilitate interactions between TFs bound to proximal or distal enhancers and the PIC bound to the core promoter. Co-repressors prevent these interactions.
1. The sequential model of activation: a cell-specific and a general co-activator are proposed to form direct interactions captured by a cohesin loop.
2. Condensates: local high concentrations of phase-separated molecules provide a regulatory mechanism to compartmentalize biochemical reactions, such as transcription.
What 3 models explain how a distal enhancer could regulate a promoter?
- Stable contact model
- Kiss and run mode: co-activators deposit PTMs at the promoter and TFs are transferred.
- Communication by diffusion model: TFs activated at the enhancer then diffuse to the promoter (likely involves condensates).
