RNA and Transcription Flashcards
What is RNA?
A nucleic acid polynucleotide formed of ribose nucleotides, contains the pyrimidine base uracil instead of thymine and is a single polynucleotide chain.
Structure of RNA
Commonly bends to form hair pin loops and coils on itself in segments, in regions of complementary strands.
what percent of the total RNA does mRNA represent?
5%
Where is mRNA located + function ?
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.
Other RNAs
tRNA, rRNA, miRNA, snRNA
tRNA size, nucleotide number and structure.
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.
tRNA function
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.
What enzyme attaches the amino acid to the tRNA molecule? + how?
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.
rRNA structure
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.
Small Nuclear RNA structure
Associated with a set of proteins, together called slurps,
SnRNA function
form the core of spiceosomes
Micro RNA structure
Small non-coding RNA molecule, formed of regions of RNA transcripts that fold back on themselves to form short hairpins
MiRNA function
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.
features of the three RNA polymerases and their main products
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
How do the RNA bases relate to the DNA coding strand?
Complementary, not identical. Adenine binds to thymine, uracil binds to adenine, cytosine binds to guanine and guanine binds to cytosine
Transcription definition
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
How does transcription begin?
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.
RNA polymerase function
Catalyses the condensation of ribonucleotides, forming phosphodiester bonds between them. Does not need a primer to bind. Works in 5’-3’ direction
How does RNA polymerase detach from the initiation complex?
TFIIH also has protein kinase activity, phosphorylates Pol II which enables it to detach
What is DNA supercoiling and why does it happen?
Conformation that DNA adopts by creating loops or coils every 10 nucleotide pairs. Due to the superhelical tension introduced by the RNA polymerase
Benefit of DNA supercoiling?
better opens up the nucleosomes
Co-transcriptional modifications
Capping, Poly A tail and splicing
What is the process of capping?
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.
Function of the cap?
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
How is the Poly A tail formed?
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.
Poly A tail function
Increases efficiency of translation and give stability to RNA
How does Poly A polymerase synthesise?
Does so without a template.
How does RNA Pol II detach?
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.
What are eukaryotic genes broken up into?
Exons-protein coding sequences, and much longer non coding introns.
RNA splicing definitions
The process of removing intron sequences from pre-mRNA to form mRNA.
What occurs in one splicing event?
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.
- 2’ OH on a particular adenosine nucleophilic attacks the exon end at an exon /intron border.
- This forms a lariat at the 3’ exon and releases the 5’ exon.
- OH on 5’ exon attacks the 3’ exon releasing the lariat.
Where does splicing take place?
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.
What performs RNA splicing?
5 small nuclear RNAs, complexed each with at least 7 protein units form the core of the spliceosome.
How is the splicing region located?
SnRNA complementary base pair to the splice junction
Splicing region structure
5’ splice site- invariant sequence of GU
branch point- adenine nucleotide involved in lariat formation
3’ splice site- invariant AG sequence
How is accuracy maintained in splicing?
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
What is alternative pre-mRNA processing?
Splicing which allows the reorganisation of axons, thus being able to form many different mRNA molecules that code for different proteins
How is alternative splicing regulated?
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.
Explain alternative polyadenylation
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
How mutations linked to pre-mRNA processing can cause disease?
- 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.
What are surveillance mechanisms?
Pathways utilised by organisms to ensure fidelity and quality of mRNA
Example of surveillance mechanism + explained
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
How are nonsense mutations recognised?
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
