L8 - RNA Transcription Flashcards
What is transcription
The process of RNA synthesis from a DNA template
Transcription is known as the
Central DOGMA of biology
What is mRNA
Messenger RNA which codes for proteins
What is the % of total RNA made up by mRNA
3-5%
What is t-RNA?
How many families?
What is found on each t-RNA?
Transfer RNA involved with translation,
49 families of tRNAs
Each carrys an amino acid and an anti-codon loop
What is rRNA
How many types
Ribosomal RNA - four main types
The main consituent
What are the functions served by non-coding RNAs
Structural and enzymatic roles within the cell
What are the four main differences seen in the primary structure of mRNA when compared to DNA
1) Ribose instead of deoxyribose
2) Uracil instead of thymine
3) Synthesised as a single strand
4) Very unstable
As being synthesised as a single strand what tends to happen to RNA
Folds on itself
What is the difference between ribose and deoxyribose
Ribose have an OH instead of a H at the 2’ position
What is the main base pairing in RNA?
What is another example of base pairing which can occur
A-U C-G
Also non Watson-Crick base pairing e.g. G-U
What secondary structures are common in RNA
Semi-loop secondary strucutres
What is meant by the tertiary strucuture
The folding of a molecule in three dimensions
What is RNA pol I involved with
Synthesis of rRNA
What is RNA pol II involved with
Transcription of protein encoding genes
What is RNA pol III involved with
tRNA - small nuclear RNA - 5s RNA
What direction is RNA always synthesised in
5’ –> 3’
What are 5 ways that RNA transcription differs from the replication of DNA
Multiple RNA polymerases bind to the same gene
No primer required
Only one strand is required as a template
Transcript does not remain bound to the template
Higher error rate than DNA replication
Where can genes be found
Can be on either side of the DNA
What is the template described as being
Anti-sense
What strand are genes found
The sense strand (but this can be either one of the strands of DNA
Unwinding how many basepairs leads to creation of supercoiling
10bp
What enzyme helps to overcome supercoiling, how
Topoisomerases
Release supercoils and allow pol2 progression
What are the four general TF elements
BRE TATA INR DPE
What protein binds to the TATA box
TATA binding protein
What general TF bindis to INR and DPE
TFIID
Where is the TATA box found in relation to the start of the gene
At the start of the gene
Where is BRE found in relation to the start of the gene
35 bp upstream
Where is INR found in relation to the start of the gene
30 bp upstream
Where is DPE found in relation to the start of the gene
30 bp downstream
What is the large complex called (this binds to other TFS
Mediator
What does an activator bind to
Enhancer element and the mediator complex
What two proteins are required to bind to the transcription initiation complex for transcritpion to begin
Chromatin remodelling factors and histone modifying enzymes
What are the three processing stages of RNA
Splicing of introns
5’ capping
Poly A tailing
What does intron splicing ahcieve
Elimination of non coding regions of mRNAs from the primary transcript to generate more mature mRNA for protein synthesis
What is 5’ capping nesc for
Stabilitiy and aids in the binding of the mRNA to the ribosome
What is poly A tailing required for
Part of the termination process (add of a poly A tail) mRNA stability
Also helps with nuclear export and translocation
What is splicing specific to
Eukaryotic mRNA
What can vary regarding splicing
Some mRNAs can be spliced in more than one way this gives rise to splice variants
Give an example of splice variation
COX3 in the brain is a splice varaint of COX1
Thought to be the target of paracetamol
What are the three sites of importantce during mRNA splicing
Splice donor site
Branch site
Splice acceptor site
What is the process of splicing (describe)
2’ OH branch site A attacks the phosphodiester bond on donor site G
Cleavage occurs at the donor site
Lariat formation
3’ OH of donor site G attacks the phosphodiester bond on the acceptor site G freeing the lariat
Lariat is the degraded
What is responsible for splicing
Spliceosome
WHat is the spliceosome
Nuclear complex made of about 150 proteins and SnRP
What is SnRNP
Small nuclear RNA and proteins
Three functions of the spiceosome
Recognition of 5’ donor and branch sites
Bring the sites together
Catlayse the RNA cleavage
What type of RNAs contain a 5’ cap
All eukaryotic
When is the 5’ cap added to the mRNA
When the mRNA is around 20-40 nucleotides long (early during synthesis)
What is the function of thr 5’ cap
Prevention of degredation of the 5’ end
What modification is made to to a base when it is 5’ capped
Guanine is methylated
Three enzymes involved in the formation of the 5’ cap
RNA triphosphatase
Guanylyl transferase
Methyl transferase
What is the function of RNA triphosphatase
Removal of Pi from the 5’ end
What is the function of guanylyl transferase
Linkage of guanine by 3 pi
What is unusual about the linkage of G in cap formatio n
By 5’ –> 5’
What is the function of methyl transferase
Addition of a methyl group to guanine
What is the poly A signal
AAUAAA
Desicribe how poly adenylation occurs to the mRNA
Cleavage site is 30-40 nucleotides downstream of the poly A signal
At the poly A signal CSTF and CPSF bind
RNA is then cleaved + release of CstF
PAP then binds to the 5’ end - poly A binding proteins bind to adenine residues
Removal of SPSF and PAP – more poly A binding protein binds
What is CSTF
Cleavage stimulating factor
What is CPSF
Cleavage and polyadenylation specific factor
What is CTD
Pol 2 C’ terminal domain
What is significant about viruses when it comes to protein synthesis
Viruses break the central dogma of DNA–>RNA–>Protein whereby reverse transcriptase enzymes can make DNA from RNA
mRNA accounts for the majority of RNA, T or F
F – RNA only accounts for between 3-5% of all RNA
How many families of tRNAs are there
49
What is the role of tRNA
tRNA participates in translation where its bound amino acid is added to the growing polypeptide chain when the tRNAs anticodon loop binds to the complimentary codon on the mature mRNA strand
Describe the role of rRNAs
Ribosomal RNAs are a major constituent of ribosomes. They are very large and very abundant and catalyse protein synthesis
What is significant about non-coding RNAs
Non-coding RNAs serve structural and enzymatic functions, acting more like proteins
What is meant by the primary sequence of RNA
The polyribonucleotide sequence
What are the key differences between RNA and DNA
RNA replaces thymine with uracil. RNA is also synthesised as a single strand and thus is unstable and rapidly degraded. Finally RNA contains a ribose sugar backbone instead of a deoxyribose sugar. The difference is an -OH group replaces the H bonded to the 2’ carbon in the sugar ring
What is meant by the secondary structure of RNAs
Secondary RNA structure refers to the base pairing that occurs within a single RNA strand.
In RNA A pairs with U and C pairs with G however it is possible for unusual base pairing such as G with U, how is this described
Non-Watson-Crick pairing
The tertiary structure of RNA refers to its interactions with other RNAs, T or F
F – tertiary structure is the RNA strands 3D conformation
Which RNA polymerase transcribes all protein coding genes
RNA Polymerase II
Which RNA polymerase transcribes all tRNAs, 5S rRNAs and other snRNAs
RNA Polymerase III
What is the role of RNA polymerase I
Transcribes rRNA genes (28S, 18S and 5.8S)
Due to internal folding within the molecule and complimentary base pairing, an RNA molecule appears as a 10th of the length of the corresponding DNA sequence, T or F
T
How is transcription different from DNA replication
Multiple RNA polymerases bind to the same gene, no primers are needed, only one strand of DNA is used as the template and polymerase only moves in one direction. Finally the transcript doesn’t remain bound to the template as in semi-conservative replication
What can be said about the error rate in transcription
Its much higher than DNA replication
Genes can be on either the sense or antisense stands of the DNA but not both, T or F
T
The RNA molecule produced in transcription will have the same sequence as the DNA sequence that codes for that gene, T or F
F – it will have the opposite sequence to the template strand
Polymerase action causes an uncoiling of the DNA that provides a force on the upstream DNA, how is this tension relived
Topoisomerases release the tension by either making single or double stranded breaks in the DNA upstream hence removing some of the coils from the superhelix
Give an example of a promoter sequence to which a general transcription factor binds to
TATA box – consisting of a TATAA/TAA/T sequence that lies 30 base pairs upstream of the coding sequence
General transcription factors are required for all gene transcription, how do they act
They act to guide RNA polymerase and bind to the promoter sequence
Specific transcription factors bind close to the promoter region, T or F
F – they bind far away
Recall the structure of a mature mRNA from start to finish
Methyl guanosine cap –> 5’ UTR –> START –> Coding Sequence –> 3’ UTR –> PolyA Tail
Which regions of pre-RNA are spliced out but only in eukaryotes
Introns
Splicing is specific to eukaryotic transcription, T or F
T
How can splicing explain how the same gene can produce different proteins when translated
The mRNA transcript can be spliced in different ways which accounts for differences in the proteins produced
Explain how splicing occurs
The 2’ OH of an adenine branch site attacks the phosphodiester bond on guanine donor site. Cleavage at the donor site results in the formation of lariat. Next a 3’ OH in the guanine donor site attacks phosphodiester bond on a guanine acceptor site freeing the lariat which is then degraded
What is the name given to the machinery that carries our RNA splicing and what does it consist of
Spliceosome – a nuclear complex made up of about 150 proteins and 5 RNAs
What is meant by SnRNPs and what do they consist of
Small nuclear ribonucleo proteins are structures that make up the spliceosome apparatus. They consist of small nuclear RNAs and proteins
What are the three functions of snRNAs
Recognise 5’ donor and branch sites, bring sites together and catalyse cleavage
Describe the synthesis of the 5’ cap in eukaryotes
The 5’ cap is present in all eukaryotic mature mRNAs. Its consists of a methyl guanosine trisphosphate cap added to the first nucleotide. The 5’-5’ linkage is unusual and provides stability to the mRNA. The 5’ cap is also required for binding of the eukaryotic initiation factors. The 5’ cap is added when the mRNA is around 20-40 nucleotides long and begins to emerge from RNA polymerase
Which end of the mRNA strand is polyadenylated
3’ end
What is the purpose of the polyA tail
The polyA tail serves as a signal to stop translation
Explain RNA polymerases role in synthesising the polyA tail
RNA polymerase contains a tail that is highly phosphorylated. The negative charge of these phosphates resembles the RNA backbone negative charge. This serves as a docking point for RNA binding proteins which bind to particular RNA sequences known as polyA signals (AAUAAA). Cleavage stimulating factor (Cstf) and cleavage/polyadenylation specific facto (CPSF) jumps off polymerase II when this sequence is detected and bind to the polyA signal and cleave the RNA. This allows PolyA polymerase to synthesise a polyA tail
PolyA tails can be up to 200 residues in length, T or F
T
