RNA and Transcription

Question 1

What is RNA?

Answer 1

A nucleic acid polynucleotide formed of ribose nucleotides, contains the pyrimidine base uracil instead of thymine and is a single polynucleotide chain.

Question 2

Structure of RNA

Answer 2

Commonly bends to form hair pin loops and coils on itself in segments, in regions of complementary strands.

Question 3

what percent of the total RNA does mRNA represent?

Answer 3

5%

Question 4

Where is mRNA located + function ?

Answer 4

Nucleus and cytoplasm.

Transmission of the genetic information from nuclear DNA to the protein synthesis site in the cytoplasm and serves as a template for the assembly of amino acids during translation.

Question 5

Other RNAs

Answer 5

tRNA, rRNA, miRNA, snRNA

Question 6

tRNA size, nucleotide number and structure.

Answer 6

25 kDa. Chain contains about 75 nucleotides.

Clover leaf shaped molecule, with an amino acid binding site at the 3’ end. Anticodon is present in the lowest lobe.

Question 7

tRNA function

Answer 7

brings amino acids to the site of protein translation, in order to bind them in the correct order and number to produce a polypeptide chain.

Question 8

What enzyme attaches the amino acid to the tRNA molecule? + how?

Answer 8

aminoacyl-tRNA synthetase. Catalyses the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate trans to form aminoacyl-tRNA.

Question 9

rRNA structure

Answer 9

Constitutes the prosthetic group of both the major and minor subunits of the nucleoproteins that form ribosomes.
Major has 3 rRNAs whilst minor has one.

Question 10

Small Nuclear RNA structure

Answer 10

Associated with a set of proteins, together called slurps,

Question 11

SnRNA function

Answer 11

form the core of spiceosomes

Question 12

Micro RNA structure

Answer 12

Small non-coding RNA molecule, formed of regions of RNA transcripts that fold back on themselves to form short hairpins

Question 13

MiRNA function

Answer 13

Role in RNA silencing. Bind to complementary regions of mRNA molecules, and cleave the mRNA, reduce length or Poly A tail, ribosomes not able to bind as well.

Question 14

features of the three RNA polymerases and their main products

Answer 14

All composed of at least 12 subunits each

RNA Polymarase I

• catalyse the linkage of ribonucleotides
• can start transcription without a primer
• contain critical Mg 2+ ion at catalytic site
• RRNA,

RNA Polymerase 2

• catalyse the linkage of ribonucleotides
• can start transcription without a primer
• contain critical Mg 2+ ion at catalytic site
• requires a set of general transcription factors to bind to correct promoter
• MRNA, SNRNA, MICRORNA

RNA Polymerase 3
- catalyse the linkage of ribonucleotides
- can start transcription without a primer
- contain critical Mg 2+ ion at catalytic site
TRNA

Question 15

How do the RNA bases relate to the DNA coding strand?

Answer 15

Complementary, not identical. Adenine binds to thymine, uracil binds to adenine, cytosine binds to guanine and guanine binds to cytosine

Question 16

Transcription definition

Answer 16

Process by which information on the strand of DNA is copied to form a new molecule of mRNA which can be used as a template in protein synthesis

Question 17

How does transcription begin?

Answer 17

RNA Pol II requires the addition of several general transcription factors that bind to the TATA sequence on the DNA. First TFIID binds, which distorts the DNA, thus acts as a landmark for the promoter.
TFIIH binds containing DNA helices, unzipping the chain. All together forms an initiation complex.

Question 18

RNA polymerase function

Answer 18

Catalyses the condensation of ribonucleotides, forming phosphodiester bonds between them. Does not need a primer to bind. Works in 5’-3’ direction

Question 19

How does RNA polymerase detach from the initiation complex?

Answer 19

TFIIH also has protein kinase activity, phosphorylates Pol II which enables it to detach

Question 20

What is DNA supercoiling and why does it happen?

Answer 20

Conformation that DNA adopts by creating loops or coils every 10 nucleotide pairs. Due to the superhelical tension introduced by the RNA polymerase

Question 21

Benefit of DNA supercoiling?

Answer 21

better opens up the nucleosomes

Question 22

Co-transcriptional modifications

Answer 22

Capping, Poly A tail and splicing

Question 23

What is the process of capping?

Answer 23

Phosphatase removes phosphate from 5’ end.

5’ end of the RNA chain is rapidly modified by the attachment of a GMP molecule, through a 5’-5’ link by guanyl transferase.

The nitrogen on guanine is then methylated.

5’ terminus is 7-methyl guanosine triphosphate.

Question 24

Function of the cap?

Answer 24

The 7-methyl guanosine triphosphate cap enables the molecule to be identified as mRNA and also preventing it from the action of phosphatase and exonuclease. Increases stability

Question 25

How is the Poly A tail formed?

Answer 25

100-200 adenine molecules added to the 3’ end of the chain.

11-30 bp before the end of the mRNA molecule, there is a poly A tail insertion signal AAUAAA.

Cleavage stimulation factor and cleavage and polyadenylation specificity factor bind.

At the AAUAAA the exonuclease cleave the RNA strand.

Poly A polymerase synthesises an additional segment of 100-200 adenine nucleotides using ATP as a donor for Adenosine units.

Question 26

Poly A tail function

Answer 26

Increases efficiency of translation and give stability to RNA

Question 27

How does Poly A polymerase synthesise?

Answer 27

Does so without a template.

Question 28

How does RNA Pol II detach?

Answer 28

3’ end of the pre-mRNA molecule is cleaved and released.

Pol II continues to transcribe, until the cleavage occurs.

The newly transcribed molecule lacks an 5’ cap, so can be rapidly degraded by exonucleases. The continued RNA degradation then allows the RNA polymerase to release its grips of the template.

Question 29

What are eukaryotic genes broken up into?

Answer 29

Exons-protein coding sequences, and much longer non coding introns.

Question 30

RNA splicing definitions

Answer 30

The process of removing intron sequences from pre-mRNA to form mRNA.

Question 31

What occurs in one splicing event?

Answer 31

Each splicing event removes one intron in two sequential processes called phosphoryl transfer reactions known as transesterifications.

These two transesterification reactions join the adjacent axons together, removing the intron as a lariat.

1. 2’ OH on a particular adenosine nucleophilic attacks the exon end at an exon /intron border.
2. This forms a lariat at the 3’ exon and releases the 5’ exon.
3. OH on 5’ exon attacks the 3’ exon releasing the lariat.

Question 32

Where does splicing take place?

Answer 32

Each splice site has a consensus nucleotide sequence that is similar from intron to intron and provides the cell with cutes for where splicing is to take place.

Question 33

What performs RNA splicing?

Answer 33

5 small nuclear RNAs, complexed each with at least 7 protein units form the core of the spliceosome.

Question 34

How is the splicing region located?

Answer 34

SnRNA complementary base pair to the splice junction

Question 35

Splicing region structure

Answer 35

5’ splice site- invariant sequence of GU
branch point- adenine nucleotide involved in lariat formation
3’ splice site- invariant AG sequence

Question 36

How is accuracy maintained in splicing?

Answer 36

Introns come out one at a time, as the pre-mRNA is transcribed preventing exon skipping.

Exon definition- the axons are quite regular in size, whereas the introns are not

Question 37

What is alternative pre-mRNA processing?

Answer 37

Splicing which allows the reorganisation of axons, thus being able to form many different mRNA molecules that code for different proteins

Question 38

How is alternative splicing regulated?

Answer 38

Associations of splicing enhancers on the mRNA will make it more likely that the spliceosome will recognise the 5’ and 3’ splice sites.

associations of splicing silencers (negative factors) will cause the spliceosome to exclude the exon.

Question 39

Explain alternative polyadenylation

Answer 39

axons have more than one polyadenylation site so a gene can code for several mRNAs that differ in their 3’ end. Therefore produce many mRNA molecules of different lengths that code for different proteins

Question 40

How mutations linked to pre-mRNA processing can cause disease?

Answer 40

• mutations at 3’ splice site can cause exon skipping
• mutations affecting splicing factors may prevent splicing
• mutations affecting the AAUAAA polyadenylation hexameter may increase cleavage of mRNA.

Question 41

What are surveillance mechanisms?

Answer 41

Pathways utilised by organisms to ensure fidelity and quality of mRNA

Question 42

Example of surveillance mechanism + explained

Answer 42

Nonsense-mediated decay - Reduces errors in gene expression by eliminating mRNA transcripts that contain premature stop codons.

If a premature stop codon is recognised, the transcript is signalled for degradation

Question 43

How are nonsense mutations recognised?

Answer 43

A normal stop codon is found in a specific terminal exon, whereas the premature stop codon is found 50-54 nucleotides upstream of the last exon-exon junction