18.01.07 Transcription mRNA processing and translation

Question 1

Where does transcription occur?

Answer 1

In the nucleus.

Question 2

What is the function of mRNA, how is it synthesised?

Answer 2

RNA polymerase II - carries genetic information

Question 3

What is the function on tRNA and how is it synthesised?

Answer 3

RNA polymerase III - Mediates decoding of mRNA sequence in conjunction with the ribosome.

Question 4

What is the function of rRNA and how is it synthesised?

Answer 4

Formation of large and small ribosome subunits. Synthesises by RNA polymerase I, with the exception of the 5S subunit synthesised by RNA polymerase III

Question 5

What is the function of the snRNAs (7SK, 7SM), how are they synthesised.

Answer 5

7SK - processing of pre-mRNA regulating transcription by controlling the positive transcription elongation factor P-TEFb

7SM - (7-2 or RNase MRP) - In mitochondria it plats a direct role in the initiation of mitochondrial DNA replication. In the nucleus it is involved in precursor rRNA processing.

RNA polymerase II (some III)

Question 6

What is the function of the scRNAs (7SL), how is it synthesised.

Answer 6

RNA polymerase III (some II)

7SL - component of the signal recognition particle (SRP). SRP directs protein trafficking in the cell.

Question 7

How is transcription initiated?

Answer 7

TF bind to the promoter region and position RNA polymerase to initiate RNA syntheses.

Question 8

Describe the structure of the promoter.

Answer 8

Promoters are cis-acting i.e. function limited to duplex on which they reside. They contain:

1) TATA box - 25bp - 35 bp from TSS defines direction of transcription.
2) GC box - ~110bp upstream from TSS. functions in either orientation.
3) CAAT box ~80bp upstream of TSS. Strongest determination of promoter efficiency. Functions in either orientation.

Question 9

What is the effect of a mutation in the TATA box?

Answer 9

Does not prevent initiation of transcription but causes transcription to begin at an incorrect location.

Question 10

What are the possible effects of mutations in the promoter region?

Answer 10

1% of single base pair substitutions caysing genetic disease occur in promoter region

Disrupt transcrtipional initation altering the amount of mRNA and protein.

Alter or abolish binding capacity of cis-acting DNA sequence motifs for trans-acting TFs.

Question 11

Give two examples of disease caused by mutations in transcriptional regulatory element.

Answer 11

1. B-thalassemia
2. FH
3. Pyruvate kinase deficiency
4. Haemophilia B

Question 12

Describe enhancers.

Answer 12

1) cis-acting short sequence elements
2) enhance transcriptional activity
3) variable distance from start site
4) function independent of orientation
5) 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

Question 13

Describe silencers.

Answer 13

1) Similar properties to enhancers

2) Inhibit transcriptional activity.

Question 14

What are the stages of post-transcription RNA processing?

Answer 14

1) splicing
2) 5’ capping
3) Polyadenylation

Question 15

When does 5’ capping occur and what is its purpose?

Answer 15

Occurs during elongation

Methylated nucleoside, 7-methylguanosine (m7G) is linked to the 5’ end of the RNA via a 5’-5’ phosphodiester bond

Possible functions:

1) Protect from 5’3’ exonuclease activity
2) Facilitate transport from the nucleus to the cytoplasm
3) Facilitate RNA splicing
4) Role in attachment of the 40S subunit of the cytoplasmic ribosomes to the mRNA

Question 16

What are the stages of 3’ polyadenylation?

Answer 16

1) AAUAAA (or AUUAAA variant) is a polyadenylation signal sequence signalling 3’ cleavage for most RNA Pol II transcripts
2) Cleavage of pre-mRNA occurs at a specific site (CA) 15-30 nucleotides downstream of AAUAAA signal
3) ~200 AMP residues are sequentially added by the enzyme poly(A)polymerase to form a poly(A) tail

Question 17

What are the possible functions of 3’ polyadenylation?

Answer 17

1) Facilitates mRNA transport to the cytoplasm
2) Stabilises some of the mRNA molecules in the cytoplasm

3) Facilitates translation
Changes in length control mRNA translation. e.g. mRNA in unfertilised eggs have short poly A tails, after fertilisation of the egg these poly A tails lengthen, activating transcription

Question 18

Summarise rRNA processing.

Answer 18

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 gene by RNA polymerase II

Question 19

Summarise the process of translation.

Answer 19

mRNA transcribed from genes in nuclear DNA 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 from the 5’ and 3’ terminal exons.

Function of UTRs - stabilise mRNA on ribosomes for translation of central segment.

Question 20

What is the effect on translation of mutations in the 5’ UTR? Give an example.

Answer 20

Alter the energy requried by the ribosome to reach the AUG and as such can lead to impatire protein synthesis.

BRCA1 has a and b promoter encoding trancripts with different 5’UTR lengths the longer is translated less efficiently. As cancerous breast tissue contains only the longer 5’UTR transcripts, BRCA1 protein expression is inhibited in breast cancer tissue as opposed to normal tissue which contains both.

Question 21

Describe the structure of ribosomes.

Answer 21

RNA-protein complexes composed of two subunits

1) 60S subunit
28S, 5.8S and 5S rRNA
~50 ribosomal proteins

2) 40S subunit
18S rRNA
>30 ribosomal proteins

Question 22

What are the three binding sites on ribosomes?

Answer 22

A site for aminoacyl-tRNA

P site for peptidyl-tRNA

E site for tRNA released following peptidyl transferase reaction

Question 23

Describe tRNAs.

Answer 23

1. 75-95nt ribonucleotides in length.
2. Mediate decoding of mRNA sequence.
3. Have specific amino acid covalently bound to acceptor arm by a specific
4. amino acyl tRNA synthetase.
5. 20 different types of tRNA synthetases (i.e. 1 specific to each amino acid)

• Synthesise the addition of a single amino acid to one or more tRNA
• Use sequence in acceptor arm to discriminate between tRNAs

6) Anti-codon sequence to recognise complimentary mRNA codon.

Question 24

What are the three stages of translation?

Answer 24

1. Initiation
2. Elongation
3. Termination

Question 25

Give an overview of mRNA translation.

Answer 25

1. mRNA translation is initiated with the binding of tRNAfmet to the P (peptidyl) site (not shown).
2. An incoming tRNA is delivered to the A (aminoacyl) site in complex with elongation factor (EF)-Tu–GTP.
3. 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.
4. 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.
5. The ribosome must then 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.
6. The ribosome is then ready for the next round of elongation.
7. The deacylated tRNA in the E site is released on binding of the next aminoacyl-tRNA to the A site.
8. Elongation ends when a stop codon is reached. Cells don’t contain tRNAs compliementary to these signals. Binding to stop codon stimulates hydrolysis of the bond between the tRNA and polypeptide at the P site.
9. Polypeptide and tRNA released, ribosomal subunits and template dissociate.

Question 26

What is the ‘wobble hypothesis’.

Answer 26

30 types of cytoplasmic tRNA and 22 types of mitochondrial tRNA

Wobble hypothesis allows interpretation of all 64 codons
- 5’ end of anticodon can bind any of several bases at 3’ end of codon