RNA Processing. Flashcards
Define cryptic splice sites?
These are nucleotide sequences that closely resemble splice sites.
What direction will a polymerase be moving if it is moving downstream?
Moving in the 5 to 3 direction.
What direction will a polymerase be moving if it is moving upstream?
Moving in the 3 to 5 direction.
Define exons?
Section of newly synthesised mRNA that will code for proteins.
Define a hexamer?
A polymer that is formed of 6 molecules.
Define introns?
Non-coding pieces of newly synthesised mRNA that are usually removed.
What are introns also known as?
As intragenic sequences.
What does a kinase enzyme do?
It adds phosphate groups.
Define a nascent RNA transcript?
The new RNA strand when synthesis has just begun and it is around 6 nucleotides long.
Define an operon?
A unit of linked genes that can be turned on or off and code for and control certain proteins.
Are operons found in eukaryotic or prokaryotic genomes??
In prokaryotic genomes.
What does a phosphatase enzyme do?
It removes phosphate groups.
What is a pyrophosphate molecule?
A molecule consisting of 2 phosphate groups.
Define RNA processing?
The molecular events that allow primary RNA transcripts to become mature RNA.
What end of RNA will an RNA polymerase add nucleotides to?
The 3 prime end.
The RNA that is produced by an RNA polymerase will be an exact copy of which DNA strand?
Of the non-template strand.
The RNA that is produced by an RNA polymerase will be complimentary to which DNA strand?
The template strand.
What is the eukaryotic process of checking the newly synthesised RNA for mistakes?
RNA processing.
What location in the cell will RNA processing take place in?
Inside the nucleus.
When will an RNA strand become known as pre-mRNA?
After processing.
When will pre-RNA become mRNA?
After 5 prime end is capped.
After a number of A’s are added to 3 prime.
After the introns have been removed and the exons have been spliced together.
What is the process of adding A’s to the 3 prime end known as?
Polyadenalation.
Does pre-mRNA also contain introns and exons?
Yes.
Are introns or exons removed from pre-RNA?
Introns.
What happens to the exons after the introns have been removed?
They are spliced together.
What happens to the mRNA once the exons have been spliced together?
The mRNA can be transported to the ribosomes and translated into proteins.
How does the ribosomes recognise the mRNA?
By the 5 prime methylguanosine cap which is located near the AUG start sequence.
Do prokaryotes use RNA processing?
Some do, but most don’t.
Why don’t prokaryotes use RNA processing?
Because the nascent RNA is used as mRNA and transcription and translation will occur at the same time.
What is the start codon on prokaryotic RNA?
AUG.
How do prokaryotic ribosomes recognise prokaryotic mRNA?
By a complimentary sequence of nucleotides called the Shine Delgarno sequence.
Where is the Shine Delgarno sequence located on prokaryotic mRNA?
At the 5 prime end of the RNA molecule.
What happens when the ribosome recognises the Shine Delgarno sequence?
It forms hydrogen bonds and will then translate the mRNA.
Will the ends of prokaryotic mRNA ever be modified?
No.
Is prokaryotic or eukaryotic mRNA said to be polycistronic?
Prokaryotic.
Why is prokaryotic mRNA is referred to as being polycistronic?
As it carries the information for several genes which will be translated into several proteins.
Do the proteins that are coded for by prokaryotic mRNA often have a related function?
Yes and are often grouped together on the chromosome.
How are prokaryotic proteins with a related function controlled?
By an operon which is a group of genes that are located together on the genome.
What is the regulatory site that is found on an operon?
A promoter which can turn the operon on and off.
What happens to the genes in an operon when the promoter is on?
The group of genes will code for protein synthesis.
What happens to the genes in an operon when the promoter is off?
None of the genes will code for protein synthesis.
Why is eukaryotic mRNA is said to be monocistronic?
Because it carries information to build 1 protein.
What proteins will introns code for?
Introns are usually regions of RNA that will not code for proteins.
The size of eukaryotic introns are often related to what?
The complexity of the organism.
What enzyme is responsible for synthesising RNA?
RNA polymerase II.
What does RNA polymerase II have on its tail?
A set of pre-MRNA processing proteins.
Where do the pre-MRNA processing proteins bind to on the RNA polymerase II?
They bind to the C terminal of the tail and they can hop off the tail when they are needed.
When is the protein that form the cap at the 5-prime end released to place the cap?
It is released when around 30 nucleotides have been formed and it will put the cap in place.
When are the splicing proteins and the proteins that add the poly-A tails released from the tail of RNA polymerase II?
At the 3 end of the RNA molecule.
When is pre-mRNA formed?
When 5 cap and the poly A tails have been added.
What 6 things does pre-mRNA consist of?
A 5 prime cap.
A 5 prime leader sequence.
Introns.
Exons.
A 3 prime leader sequence.
Poly A tails at the 3 prime end.
What are the first modifications that happen to newly synthesised pre-mRNA?
The removal of introns and then the splicing of exons to give an entire strand of mRNA that codes for a protein.
When is mature mRNA formed?
When the exons have been spliced together.
What 5 things is mature mRNA composed of?
A 5 prime cap.
A 5 prime leader sequence.
Exons.
A 3 prime leader sequence.
Poly A tails at the 3 prime end.
What kind of RNA do the 5 and 3 prime leader sequences consist of?
Non-coding RNA.
What is the job of the 5 and 3 prime leader sequences?
To guide the ribosomes to the start sequence.
What part of eukaryotic mRNA is recognised by the ribosomes?
The 5 prime cap.
What is a sign on eukaryotic mRNS that tells any degradation enzymes not to degrade it?
The 5 prime cap.
What is the first step if the addition of the 5 prime cap?
When a phosphatase enzyme removes a phosphate group from the 5 prime end.
What happens in the 2nd step of the addition of the 5 prime cap, after the phosphate group has been removed?
An enzyme uses a GTP adds a guanosine monophosphate (GMP) to the pyrophosphate group that is left at the 5 prime end.
What enzyme will add GMP to the 5 prime end when the 5 prime cap is being added?
Guanylyl transferase.
How does guanylyl transferase add GMP to the phosphate group on mRNA during the addition of the 5 prime cap?
It forms a 5 to 5 prime linkage instead a 5 to 3 prime linkage.
What is the 3rd step of the formation of the 5 prime cap on mRNA, after GMP has been added to the mRNA?
An enzyme called adds a methyl group to the 7 position of the terminal guanine on the mRNA.
Which enzyme will add the methyl group to the 7 position of the terminal guanine?
An enzyme called guanine-7-methyl transferase (G-7-MT).
What is the final step of the addition of the 5 prime cap, after the methyl has been added to the terminal guanine?
An enzyme adds a methyl group to the 2-O position on the second last base on the 5 prime end.
What enzyme adds a methyl group to the 2-O position on the second last base of mRNA during the formation of the 5 prime cap?
A 2-O-methyl transferase.
Is the 2-O methyl group is added to all eukaryotic mRNAs during formation of the methyl guanosine cap?
No.
What is the purpose of the 2-O methyl group that is added to mRNA during formation of the 5 prime cap?
To act as a self recognition site so that it is not destroyed by the immune system.
Capped RNA’s will appear exclusively on RNA’s that have been synthesised by which RNA polymerase?
RNA polymerase II and they are characteristic of eukaryotic mRNA.
What is the bond that is formed between the 5 prime cap and the primary transcript of mRNA known as?
A 5 prime to 5 prime triphosphate bridge.
What are the 3 functions of the 5 prime cap on mRNA?
To help the cell distinguish mRNA from other forms of RNA.
To prevent degradation by endonucleases.
It is where proteins will bind to when they are transporting the mature mRNA to the ribosomes.
What are the enzymes involved in the polyadenylation of the 3 prime end?
A cleavage and polyandenylation specificity factor (CPSF).
Cleavage stimulating factor F (CstF).
Poly-A polymerase (PAP).
Poly-A binding proteins (PABP).
What is the job of the cleavage and polyandenylation specificity factor (CPSF) during the polyadenylation of the 3 prime end?
It binds to the hexamer AAUAAA.
What is the job of the cleavage stimulating factor F (CstF) during polyadenylation, once the CPSF has bound to the hexamer?
It binds to a GU rich area beyond the cleavage site.
The cleavage factors will then come in and bind to the CA sequence which is at the cleavage site.
What happens to the cleavage factors after they have bound to the CA rich region at the cleavage site, during polyadenylation?
They will cleave the GU rich region from the RNA and it will be degraded.
What happens once cleavage factors have cleaved the GU rich region during polyadenylation?
Poly-A polymerase (PAP) adda around 200 A’s to the new 3 prime end.
What is the final step of polyadenylation, once the A’s have been added to the 3 prime end?
Poly-A binding proteins (PABP) bind to the poly-A tails and help to direct the RNA to the ribosomes where they will assist with translation.
What are the 2 functions of the poly-A tail on eukaryotic mRNA?
To enhance its stability by protecting it from endonuclease’s.
To regulate its transport into the cytoplasm.
What is at the sequence at the 5 prime end of an intron?
GU.
What is at the sequence at the 3 prime end of an intron?
AG.
What is the branch point sequence that is located in an intron?
It has an A located in it and it is located around 18-38 nucleotides upstream from the 3 end.
When will the removal of introns begin?
When cleavage occurs at the first intron exon junction at the 5 end.
What is the enzyme called the cleaves out introns?
A spliceosome.
What intron sequence will a spliceosome use to recognise an intron?
The GU sequence at the 5 end.
What happens to the 5 end of the cleaved intron?
The 5 end of the cleaved intron forms a loop by binding to the G from the GU to the A in the branch point sequence.
What is the loop that is formed by the 5 end of a cleaved intron known as?
The lariat structure
What bond is formed when the 5 end of a cleaved intron forms lariat strucutre?
A 2 to 5 prime phosphodiester bond.
How is the lariat structure removed from the RNA molecule?
The spliceosome cleaves the AG sequence at the 3 end which removes the entire lariat structure which will be degraded.
What happens to the exons once the introns are removed?
The spliceosomes recognise the exons and will ligate them together.
What proteins are involved in the second model for the removal of introns?
U1, U2, U4, U5 and U6 snRNP (small nuclear ribo-protein) proteins.
What is the job of the U1 and U2 snRNP proteins that are involved in the second model for the removal of introns?
They bind to the splice junctions at the 5 (GU) and 3 ends (AG).
Where will U1 and U2 bind to on the intron, in the second model for the removal of introns?
U1 will bind to the 5 prime end.
U2 will bind to the splice junction at the 5 prime end.
What happens in the the second model for the removal of introns after U1 and U2 have bound to the intron?
U4 and U6 snRNP proteins will bind to each other and will then will bind to U5 snRNP.
What happens in the the second model for the removal of introns after the U4, U6 and U5 proteins have bound?
The complex will recognise U1 and U2 and bind to them.
At this point, U4 will dissociate and its departure will form the active spliceosome.
What will the active spliceosome do when it is formed in the second model for the removal of introns?
It will cleave the 5 prime intron and form the lariat structure.
The intron is then cleaved at the 3 prime end and the exons are ligated.
Why do the methods of RNA splicing vary?
So that different species of mRNA can be produced.
What is alternative splicing?
The production of different species of mRNA
What do the different forms of mRNA that are formed by alternative splicing allow for?
For different proteins to be produced from the same gene.
How could sequence of mRNA that has 3 exons be alternatively spliced?
Exon 1 and exon 3 could be spliced together while exon 2 is trashed.
These 2 exons would produce a different protein than the 3 exons combined.
What is an example of a gene that carries out alternative splicing?
The alpha tropomyosin gene.
What are the 4 different patterns of RNA splicing?
Optional exon.
Optional intron.
Mutually exclusive exons.
Internal splice site.
What is the optional exon form of RNA splicing?
An exon may be included or excluded.
What is the optional intron form of RNA splicing?
An intron may be included in the transcript or it may be spliced out.
What is the mutually exclusive exons form of RNA splicing?
An exon may be spliced out in one transcript and a different exon may be spiced out in another.
What is the internal splice site form of RNA splicing?
A weak splice site may exist in an intron which can provide a secondary splice sight.
This is also known as intron sequence ambiguity.
What is alternative splicing controlled by?
Activators and repressor proteins.
How do repressor proteins control alternative splicing?
Repressors will bind to introns and no splicing can occur.
This is known as negative control.
How do activator proteins control alternative splicing?
Activators will bind to introns and splicing will take place.
This is known as positive control.
What process can determine whether a fruit fly is born male or female?
Alternative splicing.
What is the primary signal for determining whether a fly develops as a male or a female?
The X chromosome/autosome ratio.
If the fruit fly has a ratio of 1, how many autosomes and sex chromosomes will it have?
2 sets of X chromosomes and 2 sets of autosomes.
2/2 = 1.
If the fruit fly has a ratio of 0.5, how many autosomes and sex chromosomes will it have?
1 set of X chromosomes and 2 sets of autosomes
1/2 = 0.5.
A fruit fly with a ratio of 1 will develop into what sex?
A female.
A fruit fly with a ratio of 0.5 will develop into what sex?
A male.
What are the 3 proteins that are responsible for transmitting information about the sex ratio to cells?
SXL (sex lethal protein).
TRA (transformer protein).
DSX (double sex protein).
How does the SXL gene affect the SXL, TRA and DSX proteins?
It produces an initial alternate transcript that produces an active SXL protein.
The SXL protein affects the splicing of SXL RNA, the RNA of the transformer gene and of the doublesex gene.
How will a ratio 1 (female) affect the SXL protein?
A ratio of 1 will result in the production of a functional SXL protein as the SXL gene will be active.
What happens when a functional SXL protein is produced when female fruit flies are formed?
The SXL protein blocks a site on a piece of SXL RNA and this results in the production of more SXL proteins.
What do the SXL proteins do after they are produced during the formation of female fruit flies?
They will block a site on the TRA RNA .
What happens when SXL proteins block the TRA RNA during the formation of female fruit flies?
It results in the alternative splicing of the TRA RNA which will produce a functional TRA protein.
What happens to the functional TRA protein during the formation of female fruit flies?
It affects the DSX protein which activates a splice site on the DSX RNA.
What protein is produced from the DSX RNA during the formation of female fruit flies?
A protein that has 150 amino acids at its N terminal and 30 at its C terminal.
The 30 amino acids are female specific and will repress any male genes.
What form of the SXL protein is produced when male fruit flies are being formed?
A non-functional version of the SXL protein will be produced from the SXL gene.
What happens when the non-functional version of the SXL protein is produced during the formation of male fruit flies?
It causes a non-functional version of the TRA protein to be produced.
How does the non functional version of the TRA protein affect the DSX protein during the formation of male fruit flies?
DSX will have 150 amino acids at the C terminal and 400 at the N terminal and this will repress any female genes.
Which RNA is splice differently to produce male or female fruit flies?
The SXL RNA.
What does the pre SXL RNA consist of for the formation of fruit flies?
3 exons (2,3 and 4) and 2 introns.
How are the exons of pre SXL RNA spliced to produce a female fruit fly?
Exons 2 and 4 must be spliced in to the RNA molecule while exon 3 is spliced out.
This RNA can produce a functional SXL protein.
How are the exons of pre SXL RNA spliced to produce a male fruit fly?
All 3 exons are spliced in and no SXL protein is synthesised.
What does the pre TRA RNA consist of for the formation of fruit flies?
2 exons (1 and 2) and 1 intron.
How do a functional SXL protein and a TRA pre-mRNA interact during fruit fly formation?
A functional SXL protein binds to the TRA pre-mRNA.
This induces a cryptic splice site which causes only a small portion of exon 2 to be spliced in.
This causes the RNA will code for the production of a TRA protein.
How does TRA splicing occur without a functional SXL protein when male fruit flies are being formed?
Both exons are spliced in and no TRA protein is produced.
Will the formation of male or female fruit flies produce the TRA protein?
Female fruit flies produce the TRA protein.
What does the pre DSX RNA consist of for the formation of fruit flies?
3 exons (3, 4 and 5) and 2 introns.
How is the DSX RNA spliced to form female fruit flies?
Exon 5 is spliced out and only exons 3 and 4 are included in the RNA sequence.
This sequence will code for a female specific DSX protein.
How is the DSX RNA spliced to form male fruit flies?
Exon 4 is spliced out and the RNA consists of exons 3 and 5 which will code for a male specific DSX protein.
What is RNA editing?
The post transcriptional altering of nucleotide sequences in protein coding regions of RNA transcripts.
How can most mammals edit their RNA?
By adding or removing individual nucleotides from regions of mRNA that are not complimentary to the template strand.
What are the 3 basic stop codons used on mRNA?
UAG.
UGA.
UAA.
What organisms have extensive RNA editing?
Trypanosomes.
Which enzyme form the complimentary base pairs during RNA editing in trypanosomes?
A guide RNA (gRNA).
How do the 2 RNA strands in trypanosomes differ?
One strand of the complimentary base pairs is unedited and the other is the gRNA.
What happens during RNA editing in trypanosomes if the gRNA puts down the wrong nucleotides?
The wrong nucleotides will form a loop that extends out of the DNA strand.
What happens to the RNA loops that are formed during RNA editing in trypanosomes?
An endonuclease will cleave the loops.
What happens after the endonuclease cleaves off the DNA loops during RNA editing in trypanosomes?
An enzyme called uridyl-transferase adds U’s to the chain
This creates more nucleotides on the gRNA chain than there are on the unedited chain.
When is the mature RNA carried to the nucleus?
When the exons have been spliced together.
Which proteins will package the newly synthesised RNA?
Proteins that are involved in post transcriptional modification.
What helps the newly synthesised RNA to move to the nucleus?
Nuclear transport receptor proteins.
