Lecture 12 - Introduction to transcription Flashcards
What is the centeral dogma of molecular biology? Why do we study transcription?
Central dogma of molecular biology
Describes the flow of genetic information
1. Replication
2. Transcription
3. Translation
Why do we study transcription?
Dysregulation of transcription leads to a number of diseases such as cancer, diabetes, cardiovascular disease. Inquisitive nature, desire to know how things work.
Give an overveiw of transcription.
- RNA is transcribed in a 5’ to 3’ direction
- The first step involves recognition and binding of the promoter sequence by the RNA polymerase
- The enzyme complex separates the two strands of DNA
- Transcription initiates and the first nucleotides of the transcript are synthesised.
- Once it gets past the promoter, the complex undergoes a conformational change, stabilising its interaction with DNA
- The RNA polymerase continues elongating the RNA transcript
- Until it reaches a particular DNA sequence that causes the RNA polymerase to be released from the template
Describe Prokaryotic RNA polymerases.
RNA polymerases share common features across domains (bacteria have the simplest structure and Eukaryotes have the most complex.
Prokaryotic RNA polymerases
* Core enzyme - consisting of five polypeptides: consisting of two copies of the α subunit and one of each of β, β’, and ω.
* σ (sigma) factor - recognises the promotor and allows the RNA polymerase to bind to it
* * Holoenzyme - Core enzyme and σ factor. Analysis of promotors of many bacterial genes identifies a promoter consensus. The sigma factor binds to conserved domains on the promotor region. The similarity of the promotor sequences to the consensus correlates with the strength of the promotor. Genes that are transcribed frequently have a promotor sequence similar to the consensus. RNA polymerase has a much greater affinity for promotors of similar sequence to the consensus
Describe Eukaryotic RNA polymerases.
Eukaryotic RNA polymerases
Eukaryotic RNA polymerases require various protein factors in order to be recruited to the transcriptional start site such as TFIIA for RNA pol II. Each RNA polymerase has its own set.
1. RNA polymerase 1 (nucleolus) Transcribes rRNA (large and small ribosomal subunit) 2. RNA polymerase 2 (nucleoplasm) Transcribes mRNA (protein coding), snRNA (small nuclear RNAs found in spliceosomes) miRNA (micro RNA regulate gene expression) 3. RNA polymerase 3 (nucleoplasm) Transcribes 5S rRNA (component of large ribosomal subunit), tRNA (places amino acid in growing polypeptide chain), snRNA (small nuclear RNAs found in spliceosomes) In most eukaryotic genomes rRNA genes are arranged in tandem repeats of 1 or more chromosomes. These sites loop out from respective chromosomes and come together in a spherical mass known as the nucleolus.
Describe the formation of the Pre-initiation complex for transcription.
DNA sequences called the core promotor elements direct the assembly of the transcription initiation complex.
1. Binding of TBP to the TATA box promotes binding of TFIIB to the BRE (TFIIB Recognition Element) sequence.
TBP binds to the minor groove of DNA causing partial unwinding and DNA bending.
2. The remaining components then bind to form the pre-initiation complex.
TBP is also found in other RNA pols (tRNA, 5S RNA) due to its conserved structure.
Describe the initiation of transcription.
- All RNA polymerases open up about 14bp of duplex DNA (transcription bubble). In Pol II, the transcription bubble is opened by helicase subunits of TFIIH and, uniquely requires ATP.
- Often, RNA polymerase fails to make full length RNA on the first attempt. This ‘abortive initiation’ leads to the release of short RNAs of 2-9 nucleotides
The RNA polymerase holds the strands apart using the rudder and trigger loop (part of its structure)
There is a pore and exit channel in which newly synthesised RNA leaves and Nucleotides can enter.
Abortive initiation occurs due to the loop (Bacterial sigma factor and TGIIB in eukaryotes extending into the active site region). The loop is in a position to block the elongating transcript, so the loop must be moved in order for transcription to continue.
Displacement of the protein loop is thought to help the polymerase break away from the promotor - promotor clearance.
RNA polymerase then undergoes a conformational change that associates it very stably with DNA, and loosens its grip on initiation factors.
The biochemical reaction is catalysed by RNA pol. The α phosphate is subjected to nucleophilic attack by the 3’ OH group of the last nucleotide of the growing RNA chain.
The release of the resulting pyrophosphate, is then hydrolysed to yield monophosphate, generating energy for the reaction.
Describe mRNA processing.
- Transcriptional elongation is coupled to mRNA processing in eukaryotes
- Phosphorylation of the 5th serine in the heptad repeat on the CTD region of the RPB1 subunit occurs first
- This causes the binding of negative elongation factors that lead to transcription pausing
- Phosphorylation recruits RNA processing enzymes that add a guanosine cap to the 5’ end of the mRNA (5’ capping)
Capping leads to phosphorylation of the second serine in the CTD heptad repeat which causes the RNA pol II to resume elongation.
What is the effects of elongation and supercoililng on transcription.
As the transcription bubble continues along the DNA, positive supercoiling ahead of the polymerase and negative supercoiling increases the behind
Changes in supercoiling could causes stalling of RNA polymerase, and the tension must be relieved by topoisomerases.
In E. coli, DNA gyrase removes the positive supercoils and DNA topoisomerases I removes the negative supercoils.
How do eukaryotes overcome nucleosomes hindering eukaryotic RNA pol II.
Nucleosomes hinder eukaryotic RNA pol II
* Eukaryotes use histone chaperones to remove nucleosomes ahead of RNA polymerase, and reassemble them behind the polymerase
* Histone chaperones include ASF1, SPT6 and FACT (facilitates chromatin Transcription)
How are mistakes in transcription corrected.
- RNA polymerase stalls when it encounters obstacles or problems
- It then reverses direction and the most recently made RNA protrudes from the complex
- Transcript cleavage factors chop this off through stimulating RNA pols endonuclease activity
Transcription resumes.
How is transcription teminated in bacteria.
- Type I terminators aka Rho- independent or intrinsic terminators. No additional factors required for termination.
Type II terminators aka Rho- dependent terminators. Rho factor required for termination (uses ATP)
Describe transcriptional termination by RNA pol II (allosteric model)
- RNA pol II transcribes through polyadenylation and 3’ end processing signals
- RNA-processing complexes associate with both the processing signals and the phosphorylated CTD of the large subunit
- The mRNA is cleaved and transcription is terminated
- RNA pol II is released from the DNA
Polyadenylation and 3’ End Processing Signals: As RNA pol II transcribes a gene, it eventually reaches the termination signal. Along the DNA, specific sequences called polyadenylation and 3’ end processing signals are recognized by RNA-processing complexes.
Association of RNA-Processing Complexes: These RNA-processing complexes associate with both the polyadenylation and 3’ end processing signals on the DNA and with the phosphorylated C-terminal domain (CTD) of the large subunit of RNA pol II. The phosphorylation of the CTD plays a crucial role in coordinating the recruitment of RNA-processing factors.
Cleavage and Termination: Once the RNA-processing complexes are recruited and properly positioned, they facilitate the cleavage of the nascent RNA transcript near the polyadenylation signal. This cleavage event marks the termination of transcription.
Release of RNA Pol II: Following cleavage and termination, RNA pol II is released from the DNA template, allowing the RNA-processing machinery to complete the maturation of the mRNA transcript. The released RNA pol II can then be recycled for further rounds of transcription.
Describe transcriptional termination by RNA pol II (torpedo model)
- The mRNA is cleaved at the poly(A) site.
- Nascent RNA downstream of the poly(A) cleavage site is digested by a 5’ to 3’ ribonuclease (rat1)
Polymerisation is disrupted and RNA pol II dissociates from the DNA template.
- Nascent RNA downstream of the poly(A) cleavage site is digested by a 5’ to 3’ ribonuclease (rat1)
