19.01.07 Transcription, mRNA processing (excluding splicing) and translation Flashcards
Transcription, mRNA processing (excluding splicing) and translation
Central dogma
The expression of genetic information in a cell is almost exclusively unidirectional. DNA>RNA>protein
Where does transcription occur
Nucleus (limited extent in mitochondrial)
What direction is RNA synthesised?
5’ to 3’, complimentary to template (anti sense strand) with same bases except U/T.
how many classes of RNA polymerase molcules
3 (I, II, III)
how does transcription start
-Transcription factors bind to the promoter region and position RNA polymerase to initiate RNA synthesis.
Are transcription factors trans- acting?
Yes, migrate to sites of action following synthesis by remotely located genes
Are promoters trans-acting
No, they are cis-acting. Function is limited to the duplex on which they reside.
What other elements are cis-acting
Other than promoters, enhancers and silencers are also cis-acting
Elements of promoters
TATA box, GC box, CAAT box
TATA box
TATAAA sequence. 25-35 bp upstream of transcriptional start site. Able to define direction of transcription, indicates the DNA strand to be read. Mutations can cause transcription to begin at an incorrect location
GC box
Variant of GGGCGG. 110bp upstream of transcriptional site. Can function in both directions.
CAAT box
GGCCAATCT. 80bps upstream. Strongest determination of promoter efficiency. Functions in both directions
Mutations in promoters
1% of single base pair substitutions causing genetic disease occur in promoter regions. Disrupt transcription initiation (alter/abolish cis-activing DNA sequence motids for trans acting TFs), leading to altered amounts of mRNA and thus protein.
enhancers
Enhance transcriptional activity. Variable distrance from start site. Function independent of orientation. Cause DNA between promotor and enhancer to loop out.
silencer
inhibit transcriptional activity
Main steps of RNA processing
5’ capping, 3’ polyadenylation
5’ capping, when does it occur
During elongation
functions of 5’ capping
protect from 5’ - 3’ exonuclease activity. Facilitate transport from nucleus to cytoplasm, facilitate RNA splicing. Attaches 40s subunity of ribosome to mRNA
What happens during 5’ capping
methylated nucleoside, m7g (7-methylguanosine), is linked to the 5’ end of RNA by a phosphodiester bond.
3’ polyadenylation
AAUAAA (or AUUAAA) signal sequence is sequentially added by pol(A)polymerase, around 200 added, to form a poly(A) tail. Acts as a signal for 3’ cleavage for RNA pol II transcripts
Functions of 3’ polyadenylation
Facilitates mRNA transport to cytoplasm, stabilises mRNA molecules, facilitates translation
Mutations affecting 3’polyadenylation
rare. Causes read0-through and reduced accumulation of alpha-haemoglobin in haemoglobin H (HbH) disease.
rRNA processing
RNA polymerase I makes a large pre-rRNA molecule. Then cut into 3 pieces: 18S, 5.8S and 28S
What makes 5S (smallest eukaryote rRNA
RNA polymerase III
Translation
mRNA transcribed from genes in nuclear DNA, migrates to cytoplasm and engages with ribosomes, tRNA and other components to direct polypeptide synthesis.
Functions of untranslated regions (UTRs)
Stabilise mRNA on ribosomes for translation of the central segment
Mutation in 5’UTR
BRCA1 has an alpha and beta promoter which encode transcripts with different 5’UTR lengths. The longer is translated less efficiently. Cancerous breast tissue has the longer 5’UTR transcript so BRCA1 protein expression is inhibited in cancer tissue.
Ribosomes
RNA-protein complexes
Structure of a ribosome
2 subunits: 60S and 40S subunits.
Ribosome 60s composed of
28S, 5.8S and 5S rRNAs, with various ribosomal proteins (50)
Ribosome 40s composed of
18S rRNA and >30 ribosomal proteins
How many ribosome biding sites
- A site (aminoacyl-tRNA), P site (peptidyl- tRNA), E site (for tRNA released following peptidyl transferase reaction)
What is tRNA
75-95 nt ribonucleotides in length
What do tRNAs do
Mediate decoding of mRNA sequence. Have a specific amino acid covalently bound to acceptor arm by amino acyl tRNA synthetases
How many types of tRNA synthetases
20 (1 specific to each amino acid).
What does the tRNA acceptor arm contain
Sequence to allow discrimitation of tRNAs by tRNA synthetases.
tRNA anti codon sequence
This recognises the complimentary mRNA codon.
Steps of translation
Initiation, elongation, termination
What initiates mRNA translation
Binding of tRNAfmet to P (peptidyl) site.
What is then delivered to A (aminoacyl site)
tRNA in complex with elongation factor= EF-Tu-GTP
What happens when codon-anticodon pairing occurs
Activates GTPase centre of ribosome, causing hydrolysis of GTP and the release of aminoacyl end of tRNA from EF-TU.
What also happens when tRNA binds
COnformation changes in rRNA, optimally orientates the peptidyl-tRNA and aminoacyl-tRNA for the peptidyl-transferase reaction to occur.
What happens during the peptidyl-transferase reaction
transfer of the peptide chain onto the A-site tRNA
How does the ribosome shift in the 3’ mRNA direction
GTPase EF-G casuses 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.
When is the deacylated tRNA in the E site released
Upon binding of the next aminoacyl-tRNA to the A site.
When does elongation end
when stop codon is reached. No tRNAs complimentary to the stop codon. Leads to hydrolysis of the bond between tRNA and polypeptide at the P site. They are then released and ribosomal subunits and template dissociate
How many combinations of codons for amino acids
64 possible codons (4 bases at 3 positions, 4^3) with 20 amino acids
Wobble hypothesis
allows interpretation of all 64 codons. 5’ end of anticodon can bind any of the several bases at 3’ end of codon.
When does nuclear-encoded mRNA translation stop
When a termination codon is encountered (UAA, UAG, UGA)
