Transcription, mRNA processing (excluding splicing) and translation Flashcards
What is transcription?
Occurs in the nucleus (mtDNA genes are transcribed in the mitochondria).
RNA is synthesised in a 5’à3’ direction as a single strand, complimentary to the template (antisense) strand and has the same base sequence (except U/T) as the non-template (sense) strand.
There are 3 different classes of RNA polymerase, the majority of protein coding genes use RNA polymerase II
How is transcription initiated?
Transcription factors bind to the promoter region and position the RNA polymerase to initiate RNA synthesis (basal transcription apparatus = polymerase + all of its associated general transcription factors).
Transcription factors are trans-acting i.e. migrate to sites of action following synthesis by remotely located genes
Promoters are cis-acting i.e. function limited to DNA molecule on which they reside
Give some examples of promoters
TATA box
Often TATAAA or variant
25-35bp upstream from the transcriptional start site
Able to define direction of transcription and indicates the DNA strand to be read
GC box
A variant of GGGCGG
~110 bases upstream from the transcriptional start site
Found in a variety of genes, many lacking a TATA box (inc. house-keeping genes)
Functions in either orientation
CAAT box GGCCAATCT or variant ~80bps upstream of start site Strongest determination of promoter efficiency Functions in either orientation
What are the properties of enhancers?
cis-acting short sequence elements
enhance transcriptional activity
variable distance from start site
function independent of orientation
bind gene regulatory proteins causing DNA between promotor and enhancer to loop out and proteins bound to enhancer to interact with promoter bound TFs or RNA polymerase
play a role in controlling the specificity of gene expression
What are the properties of silencers?
Similar properties to enhancers
Inhibit transcriptional activity
Give some examples of cis and trans regulatory elements in human disease
1% of single base pair substitutions causing genetic disease occur in promoter region, disrupting transcriptional initiation altering amount of mRNA and protein e.g. LDLR promoter resulting in familial hypercholesterolaemia
Fragile X full expansion (>200 rCGG repeats) in the 5’UTR of FMR1 causes aberrant gene methylation and promoter silencing resulting in the Fragile X phenotype
Pathogenic variants in the SHH enhancer, ZRS, result in polydactyly
Topologically associating domains (TADs) are regions of the genome with high chromatin interactions, separated by regions with fewer domains. Disruption of TAD boundaries can lead to ectopic enhancer-promoter interactions, leading to altered gene expression e.g. limb malformations in patients with structural variation at the EPHA4 locus.
Pathogenic variants in transcription factors can affect expression of their target genes e.g. GATA family of transcription factors are regulators of gene expression in haematopoietic cells and can cause predisposition to myelodysplastic syndromes and acute myeloid leukemia.
What is 5’ Capping of the RNA transcript?
Occurs shortly after the initiation of transcription
Methylated nucleoside, 7-methylguanosine (m7G) is linked to the 5’ end of the RNA via a 5’-5’ phosphodiester bond
Possible functions:
Protect from 5’à3’ exonuclease activity
Facilitate transport from the nucleus to the cytoplasm
Facilitate RNA splicing
Role in attachment of the 40S subunit of the cytoplasmic ribosomes to the mRNA during translation
What is 3’ Polyadenylation of the RNA transcript?
AAUAAA (or AUUAAA variant) is a polyadenylation signal sequence signalling 3’ cleavage for most RNA Pol II transcripts
Cleavage of pre-mRNA occurs at a specific site (CA) 15-30 nucleotides downstream of AAUAAA signal
~200 adenylate (AMP) residues are sequentially added by the enzyme poly(A)polymerase to form a poly(A) tail
Possible functions:
Facilitates mRNA transport to the cytoplasm
Stabilises some of the mRNA molecules in the cytoplasm
Facilitates translation
rRNA processing
4 main eukaryotic rRNAs: 28S, 18S, 5.8S and 5S; all are components of the large subunit of the ribosome, with the exception of 18S rRNA which is found in the small subunit.
RNA polymerase I makes a large RNA molecule called pre-rRNA (45S) which is subsequently cut into three pieces yielding the 28S, 18S, and 5.8S rRNA molecules.
The smallest eukaryote rRNA (5S) is made from a separate cluster of genes by RNA polymerase III.
mRNA translation
mRNA transcribed from nuclear DNA genes migrates to the cytoplasm and engages with ribosomes, tRNA and other components to direct polypeptide synthesis.
Only the central segment of a typical mRNA molecule is translated.
5’ and 3’ untranslated regions (UTRs) are transcribed and assist with binding (Kozak box) and stabilising the mRNA on ribosomes and for efficient translation; UTRs themselves are not translated.
Also contain cis-regulatory elements which regulate the level of translation
Transfer RNA (tRNA) properties?
75-95nt ribonucleotides in length.
Mediate decoding of mRNA sequence à protein
Anti-codon loop recognises complimentary mRNA codon; amino acid is covalently linked to 3’ OH group.
Amino acid attached by specific amino acyl tRNA synthetase (20 different types).
Synthesise the addition of a single amino acid to one or more tRNA
Use sequence in acceptor arm to discriminate between tRNAs
Amino acid – tRNA complex = aminoacyl tRNA
Only the first 2 nucleotides in the anticodon strictly follow Watson-Crick base pairing rules and create codon specificity, whereas the 3rd base is known as the ‘wobble base’ where the rules are more relaxed.
This is because the genetic code is degenerate. (4=bases)3=base positions in codon=64 possible codons with 20 amino acids
30 types of cytoplasmic tRNA and 22 types of mitochondrial tRNA
Errors in translation and association with human disease
Changes in the 5’ UTR can impair protein synthesis and cause human disease e.g. BRCA1 has α and β promoter encoding transcripts with different 5’UTR lengths, the longer of which is translated less efficiently (more energy is required by the ribosome to reach AUG). As cancerous breast tissue contains only the longer 5’UTR transcript, BRCA1 protein expression is inhibited in breast cancer tissue as opposed to normal tissue which contains both.
Pathogenic variants affecting the termination codon, polyadenylation signal and secondary structure of 3′-UTR of mRNA can cause translation deregulation and disease e.g. Mytonic dystrophy (DM1) is caused by expansion of a triplet repeat in the 3’UTR which has a toxic gain of function effect.
Several pathogenic mitochondrial tRNA variants cause human disease e.g. m.3243A>G tRNALeu(UUR) which most commonly results in MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like Episodes)
Pathogenic variants in tRNA synthetase genes have been reported in association with autosomal recessive and dominant disorders e.g. YARS (tyrosyl-tRNA synthetase) and AD Charcot-Marie-Tooth
Translation initiation
5’ cap of mRNA recognised by the small ribosomal subunit – which then binds to mRNA and scans along the 5’UTR until start codon identified
Inititator tRNAMet pairs with the AUG start codon and binds to the P (peptidyl) site of the ribosome
The appropriate aminoacyl tRNA is delivered to the A (aminoacyl) site of the ribosome in a complex with elongation faction (EF)-Tu-GTP.
Correct codon–anticodon pairing activates the GTPase centre of the ribosome, which causes hydrolysis of GTP and release of the aminoacyl end of the tRNA from EF-Tu.
Binding of tRNA also induces conformational changes in ribosomal (r)RNA that optimally orientates the peptidyl-tRNA and aminoacyl-tRNA for the peptidyl-transferase reaction to occur, which involves the transfer of the peptide chain onto the A-site tRNA.
Translation elongation
The ribosome must shift in the 3’ mRNA direction so that it can decode the next mRNA codon. Translocation of the tRNAs and mRNA is facilitated by binding of the GTPase EF-G, which causes the deacylated tRNA at the P site to move to the E (Exit) site and the peptidyl-tRNA at the A site to move to the P site upon GTP hydrolysis. The ribosome is ready for the next round of elongation.
The deacylated tRNA in the E site is released on binding of the next aminoacyl-tRNA to the A site.
Translation termination
Elongation ends when a stop codon is reached (UAA, UAG, UGA or in mammalian mitochondria UAA, UAG, AGA, AGG). Cells don’t contain tRNAs complimentary to these signals. Binding to stop codon stimulates hydrolysis of the bond between the tRNA and polypeptide at the P site.
Polypeptide and tRNA released, ribosomal subunits and template dissociate.
