Flashcards in Control of gene expression (transcription) Deck (53):
What are the names given to the 2 strand of DNA during transcription?
- Coding/sense strand: Strand of DNA with the same base sequence as the synthesised RNA molecule (aside from T → U transitions). This is the non-template strand.
- Non-coding/antisense strand: Strand of DNA with complementary base sequence to synthesised RNA molecule. This is the template strand.
Why are copying errors frequent in RNA?
RNA polymerases don't have proofreading or intrinsic 3'-5' exonulcease activity.
Why are copying errors unimportant in transcription?
1. Repeated transcription of gene.
2. High redundancy of genetic code.
3. Single amino acid substitution usually makes little difference to the structure/function of proteins.
What are the 2 forms of RNA polymerases?
1. Core enzyme: 2 x α, β, β'
2. Holoenzyme: 2 x α, β, β', σ
What are the differences between eukaryotic and prokaryotic transcription?
- Prokaryotic DNA is not found in nucleus, while eukaryotic DNA is, so transcription and translation are separated in eukaryotes.
- Prokaryotic DNA does not contain introns.
- Prokaryotic DNA is often transcribed in operons.
- Prokarytoic DNA is often simultaneously translated and transcribed.
- Eukaryotes have wider range of promoters than prokaryotes.
What is the significance of the differences between eukaryotic and prokaryotic transcription?
Prokaryotic transcription is much faster than eukaryotic transcription.
What is the process of transcription?
1. RNAP binds to the DNA via the initiation site of the gene to be transcribed.
2. The double helix is melted and an unwound region called the transcription bubble is formed (fig. 2).
3. RNAP catalyses the joining of NTPs onto the growing RNA sequence using the template strand of DNA as template.
4. Once the DNA has been transcribed, it reforms double helix. This allows the transcription bubble to move along the DNA molecule.
5. RNAP is processive and RNA synthesis is continuous. The RNAP molecules achieve this processivity in the absence of any clamp structures.
6. When RNAP reaches the termination site, it drops off and transcription is complete.
What are the 2 consensus sequences of a prokaryotic promoter?
1. Pribnow (-10) box: TATAAT
2 -35 box
What is the function of σ factors?
Determines specificity of transcription machinery.
What is the function of the consensus sequences of prokaryotic promoters?
The consensus sequences for each gene are unique and form the binding sites for specific σ factors.
What ensures that transcription only occurs in 1 direction from promoter?
Asymmetrical nature of the promoter and RNA polymerase holoenzyme.
What are the methods of prokaryotic transcription termination?
1. Rho independent
2. Rho dependent
At what site on DNA does rho factor bind to?
C-rich sequence preceeding the GC hairpin
What are the ways prokaryotic transcription is regulated?
1. Promoter efficiency
2. Availability of σ factor
3. Transcription factors (activators and repressors)
What types of transcriptional events are there?
- Constitutive: Occur in all cells (non-specific)
- Inducible: Occur in response to specific stimuli
- Cell/tissue specific: Occur in specific tissue types only
- Developmental: Occur at specific stages of development only
What are the types of eukaryotic RNAPs?
- RNAPI: Pre-rRNA
- RNAPII: Pre-mRNA, miRNA
- RNAPIII: tRNA
What are DNA promoters?
Sequences of DNA in the vicinity of the initiation point that bind to TFs involved in initiating transcription.
What are regulatory elements?
Sequences of DNA that bind TFs involved in transcriptional regulation of the expression of genes.
What is the consensus sequence for the eukaryotic promoter?
TATA box (-25bp)
What is the core promoter element?
Sequence of DNA in promoter region that bind to general transcription factors (GTFs) that are required for initiation of transcription.
What is the sequence of events of pre-initiation complex (PIC) formation?
1. TFIID (containing TATA binding protein) binds to the promoter.
2. TFIIA and TFIIB are then recruited by TFIID.
3. TFIIF then recruited with the complex, bringing RNAPII with it.
4. TFIIE and TFIIH are recruited to the complex.
5. Interaction of GTFs with RNAPII activates it and initiates transcription.
What are upstream promoter elements?
DNA sequences that form binding sites for additional TFs required for transcriptional initiation that are specific to the gene.
How can one PIC initiate multiple transcriptional events?
Parts of the PIC remains after transcription initiated, which can recruit additional RNAPs.
How can histones be modified in transcriptional regulation?
- Acetylation (on Lys residues by histone acetylases)
How can TFs be controlled?
What are the DNA binding domains on TFs?
- Basic helix-loop-helix (bHLH)
- Nuclear hormone receptor DNA-binding domain (steroid receptors)
- Winged helix (e.g. p.53)
How do TFs regulate transcription?
- Recruit GTFs
- Direct interactions with RNAP
- Recruits co-activators that influence DNA packaging
What are the differences between enhancers and promoters?
- Enhancers are not located in close vicinity to gene
- Enhancers are not specific to a certain gene
How do enhancers function?
By interacting with PIC and promoting the binding/activity of RNAPII.
How are they able to function over great distances?
What are the stimuli that response elements respond to?
- Heat shock
- Heavy metals
- Steroid hormones
How does CREB function?
1. Activation of adenylate cyclase pathway (e.g. glucagon) results in the activation of PKA.
2. PKA phosphorylates CREB which is bond to protein.
3. Phosphorylated CREB recruits CBP which directly interacts with PIC and has histone acetylase activity.
What is the structure of steroid response elements?
- Hormone binding region
- DNA binding region
- Variable region
How does the glucocorticoid receptor (GR) work?
1. Glucocorticoid binds to GR.
2. GR dissociated from HSP90.
3. GR dimerises and binds to glucocorticoid response element (GRE), promoting transcription by chromatin modifications.
What are the functions of capping?
- Protects RNA from 5' exonuclease activity
- Aids in transport out of the nucleus
- Aids in translational initiation
- Increases efficiency of splicing
- Allows coding mRNA to be distinguished from fragmented junk RNA.
What is the structure of eukaryotic cap?
1. γ phosphate on 5' nucleotide removed
2. GMP jointed onto the β phosphate via 5' phosphate.
3. Guanisine residue methylated on carbon 7 by enzyme 7-guanyl-methyltransferase.
4. If 5' carbon is A residue, 2' carbon may also be methylated.
What is the function of the poly-A tail?
- Aids in transport out of the nucleus
- Protects against 3' exonucelase activity and defines lifespan of mRNA.
- Defines 3' terminus of RNA transcript
What is the process of poly-A tail formation?
1. Specific endonuclease recognises the consensus sequence (AAUAAA), which signifies the end of the gene.
2. mRNA is cleaved just distal to the consensus sequence, proximal to GU/U-rich sequence.
3. Poly(A) polymerase catalyses the addition of the poly(A) tail onto the 3’ end of the mRNA, which is activated by binding of the CPSF.
What is the experimental use of poly-A tails?
Poly-T DNA fragments can bind to poly-A tails. Reverse transcriptase and DNA polymerase then used to make double-strand cDNA.
Which genes do not contain introns?
What factors are required to mediates splicing?
- snRNPs (small nuclear ribonucleoproteins).
- uridine-rich RNA molecules.
- Form spiceosomes.
What is the consensue sequence of splice sites?
What are the sequence of events that occur during splicing?
1. U1 binds to the 5’ splice site of the intron.
2. U2 binds to the polypyrimidine tract found between branch point and AG at 3’ end.
3. 2’OH of branch point A residue attacks phosphate on 5’ intron nucleotide.
4. A ‘lariat’ structure is formed between the 5’ nucleotide and branch point A residue.
5. 3’OH on 5’ nucleotide attacks phosphate on 3’ nucleotide, breaking the phosphodiester bond between the 3’ nucleotide and exon, excising the intron.
6. Exons are ligated.
7. Lariat excised and broken down.
What is significant about catalysis of splicing events?
They are catalysed by RNA in the snRNPs, so are examples of ribozyme catalysis.
What is the structure of mature mRNA?
2. Coding sequence (Open reading frame)
What types of mutations cause splicing errors?
Mutations in the splice site consensus sequences result in spliceosomes binding to so-called cryptic splice sites. This would cause splicing errors leading to disease.
How can alternative splicing cause different proteins to be generated?
1. Exons are skipped.
2. Introns are skipped.
3. Premature stop codons are introduced.
What mediates alternative splicing?
- SR proteins that bind to exonic splicing enhancers (ESEs).
- Heterogeneous nuclear RNPs (hnRNPs) that bind to exonic/intronic splicing silencers (ESSs/ISSs).
What is the clinical significance of alternative splicing?
- Wilms’ tumour is kidney tumour caused by mutation in the WT1 gene, a tumour suppressor, caused by errors in alternative splicing. This causes formation of deformed WT1 protein (e.g. truncation of DNA-binding domain (N-terminus)/activation domain (C-terminus)), leading to cancer.
- Frasier syndrome is caused by alternative splicing-related mutations that maintain normal production of WT1, but in a 1:2 KTS +/- ratio as opposed to a 2:1 KTS +/- ratio.
What are possible RNA editing mechanisms?
1. Methylation of A residues (not technically RNA editing).
2. Base modifications (e.g. hydrolytic deamination in C → U or A → I (Inosine, read as G) conversion).
What is the clinical significance of RNA editing?
Neurofibromastosis (NF1) is a cancer of the CNS/PNS. It is caused by a C → U conversion resulting in the formation of a premature stop codon in the RNA transcript of the NF1 gene (whose gene product is a tumour suppressor). This results in truncation of the GAP domain (which regulates RAS) and inactivation of the protein.
What is the process of RNA degradation?
1. Shortening poly(A) tails.
2. Removal of 5’ cap.
3. Presence of specific binding sites on mRNA (AU-rich elements, ARE) marking it for destruction (e.g. c-fos).