Transcription Pt. 2 Flashcards
What needs to happen to the mRNA transcript after transcription but before translation in eukaryotes?
processing
What needs to happen to the mRNA transcript after transcription but before translation?
processing
During the elongation stage, what does the C-terminal tail of RNA Polymerase II do? Why?
recruit enzymes so that as the new mRNA is produced, processing can begin right as it leaves RNAPII
T or F: Processing is completely separate from transcription - why/why not?
False
Processing is continuous with transcription because the enzymes recruited during elongation start processing the new mRNA transcript as it leaves RNAPII
Describe nucleases
Enzymes that cut DNA or RNA
What are the two kinds of nucleases?
exonuclease
endonuclease
Describe exonuclease
enzymes that cut DNA or RNA at either the exposed 5’ or 3’ end of a strand
Describe endonucleases
enzymes that cut DNA or RNA within the strand
How do our cells defend against viruses? What is the issue with this?
by making lots of RNA exonucleases that can cut up viral RNA
it is good cause it keeps us healthy, but it means the mRNA is vulnerable to degradation by our own nucleases
Why is the cytosol a dangerous place?
because of the nuclease enzymes that can cut up DNA or RNA
What is one main reason processing required?
To make the mRNA less vulnerable to exonucleases in the cytosol so it can be translated
What are the 3 major mRNA processing steps?
Addition of 5’ methylated guanine nucleotide cap
Addition of Poly-A tail at 3’
RNA splicing
Where do all 3 major mRNA processing steps occur?
in the nucleus
In relation to the RNA exiting the RNAPII, when do the 3 major mRNA processing steps happen?
While the transcript is leaving RNAP II (co-transcriptionally)
Describe how the 5’ cap is added
When the first 25 nucleotides are produced and the mRNA is emerging from RNAPII, capping factor enzymes bind to the 5’ end of mRNA
To add the 5’ cap, what is added to the 5’ end of mRNA?
capping factor enzymes
What were the capping factor enzymes previously attached to?
the C-terminal RNAPII tail
Which 3 capping enzymes are required to add the 5’ cap?
phosphatase
guanyl transferase
methyl transferase
Describe phosphatase and its function
a capping factor enzyme required for adding the 5’ cap onto mRNA because it removes a phosphate from the 5’ end
Describe guanyl transferase and its function
A capping factor enzyme that is required for the addition of the 5’ cap because it adds an inverted GMP to the 5’ end
Describe methyl transferase and its function
A capping factor enzyme that is required for the addition of a 5’ cap onto the end of mRNA because it methylates the GMP
Briefly describe the steps of adding the 5’ cap onto mRNA
- phosphatase cleaves one of the phosphates from the 5’ end of mRNA
- guanyl transferase adds an inverted GMP to the 5’ end
- methyl transferase adds a methyl group to the incorporated GMP
What is the final product of the addition of the 5’ cap?
a 7-methylguanosine molecule connected to the 5’ end of the mRNA by 3 phosphates
How is the 7-methylguanosine molecule connected to the 5’ end of mRNA?
a 5’-5’ triphosphate linkage
Describe a 5’-5’ triphosphate linkage
the linkage created by the 3 phosphates that bond to the 5C of the sugar on the methylguanosine and the 5C of the sugar on the mRNA
What is the addition of the poly-A tail coupled with?
termination
What are the 2 basic steps of adding a poly-A tail?
an endonuclease cleaves the RNA transcript after the AAUAAA sequence
a poly-A polymerase adds a poly-adenosine tail of ~200-250 adenosine bases after the cleave site
Does poly-A polymerase use a template to add the adenosine bases when it creates the poly-A tail?
No
What are the cap and the tail of mRNA?
cap: 7-methylguanosine molecule bonded to the 5’ mRNA end by a 5’-5’ triphosphate linkage
tail: a poly-adenosine tail of ~200-250 adenosine bases linked to the 3’ end of mRNA
What are the functions of the head and tail of mRNA?
protect mRNA against exonucleases (degradation) in the cytosol
aid in recognition by nuclear export proteins
aid in recognition by the ribosome
aid in proper positioning of the mRNA on the ribosome for protein synthesis
T or F: an mRNA in the cytosol can exist long enough to bind to the ribosome and be translated properly without a head and tail - why/why not?
False
the head and the tail protect the mRNA from being degraded by the exonucleases in the cytosol
in 1977, what did experiments with a DNA/single-stranded eukaryotic processed mRNA hybrid show? What did this mean?
unhybridized loops in the DNA
Meant the DNA sequences coding for the final mRNA were not continuously colinear = final mRNA is interrupted
Describe introns
Sequences of RNA in the coding region that are removed during processing
What are the loops seen in electron micrographs when processed mRNA is used in an experiment? what does this suggest?
parts of the DNA nucleotide sequence that do not have any complementary RNA
pieces of RNA in the coding region are removed during processing
Describe exons
Sequences of RNA that are in the final mRNA
EXpressed sequences
Are exons present after processing?
YEs
What is removed from the RNA during processing?
introns = intervening sequences of RNA
How are introns removed?
breaks in the RNA at the intron/exon boundaries = splice sites
What does a mature mRNA transcript consist of?
the 5’ cap
the poly-A tail
removed introns
What is the mRNA transcript called before processing is complete?
pre-mRNA
T or F: pre-mRNA can leave the nucleus
False
only mature RNA can leave the nucleus because pre-mRNA will not be recognized by exportins (does not have a nuclear export signal)
Describe mRNA splicing
the process of intron removal and exon ligation that joins all the exons into one RNA transcript
When does mRNA splicing occur?
continuously with RNA synthesis
What causes mRNA splicing to occur?
a spliceosome catalyzes the reaction
Describe a spliceosome
a large complex of RNA and protein used for splicing
it is composed of snRNPs
What are the components of a spliceosome?
snRNPs = 5 snRNAs + complex of protein subunits
What is an snRNA?
noncoding RNA in the spliceosome
small nuclear RNA
What does it mean for RNA to be ‘non-coding’?
it has a function other than making proteins
What are the 5 snRNA molecules in a spliceosome?
U1 U2 U4 U5 U6
What is each snRNA complexed with in the spliceosome?
multiple proteins to form a snRNP
What is a snRNP?
small nuclear ribonucleoprotein
the 5 snRNAs and their complexes of protein subunits make up an snRNP
What does an snRNP consist of?
the 5 snRNAs (U1, U2, U4, U5, U6) and their complexes of protein subunits
What is the function of the snRNA in mRNA splicing?
it catalyzes the mRNA at the splice sites
T or F: snRNA exists by itself
FALSE it only exists in complex with proteins
What 3 things does every intron have?
a GU sequence at the 5’ end
an AG sequence at the 3’ end
A single Adenine nucleotide in a particular spot in the middle (branch point)
What is a branch point?
The specific spot in the middle of an intron where a single adenine nucleotide is located
What is the purpose of the GU, AG, and A positions on an intron?
to help recognize the intron boundaries
to recruit the spliceosome
and to properly splice out the intron
How does a spliceosome form?
in steps as the different snRNPs are added one by one to the splice sites
What is the first step of spliceosome activity?
U1 snRNP binds to the GU at 5’ of intron
What is the 2nd step of spliceosome activity? (after U1 binds)
U2 snRNP binds to the adenine in the middle of the intron
What happens after both U1 and U2 have bonded to the intron?
the rest of the snRNPs (U4, U5, U6) are recruited together which fold the pre-mRNA into a loop
at which point all 5 of the snRNPs are bound to the intron
What does the binding of the U4-U6 snRNPs to the intron cause?
the pre-mRNA to fold into a loop
T or F: when the pre-mRNA forms a loop, only the U4, U5, and U6 snRNPs are bound to the intron
False
All 5 of the snRNPs are bound to the intron at this point
What happens after the loop is formed?
The entire complex rearranges and displaces U1 and U4
What is the purpose of displacing U1 and U4 from the spliceosome complex?
to expose the catalytic snRNA at U6 to cleave the intron
Where does the spliceosome cut the mRNA? how does it cut it?
at the 5’ GU (beginning of the intron) with endonuclease activity catalyzed by the snRNA at U6 of the spliceosome
What happens to the new 5’ end of RNA after the mRNA was cleaved by endonuclease?
the 5’ end covalently bonds to the branch point adenine to create a lariat
What is a lariat?
a lasso/loop shape of RNA created when the mRNA is spliced by an endonuclease and the new 5’ end covalently bonds to the branch point adenine
How is the lariat released? What else goes with it?
the spliceosome complex cleaves the 3’ AG after the lariat forms and it is released with the spliceosome
What happens to the mRNA when the spliceosome and lariat are released from the mRNA?
2 exons are ligated
What happens to the intron lariat after it released from the mRNA?
it is degraded
What happens to the spliceosome complex (snRNPs) after the intron is spliced?
it is released and the snRNPs are recycled to begin the process again at another intron
List the basic splicing steps (8)
- intron is transcribed and emerges from RNAPII
- U1 binds to 5’ GU
- U2 binds to A branch point
- U4, U5, U6 recruited
- snRNP rearranges and displaces U1 and U4 to expose catalytic snRNA site in U6
- 5’ GU cleaved and joined to branch point A to form lariat
- 3’ AG cleaved, lariat released, spliceosome complex released, 2 adjacent exons ligated
- lariate intron is degraded and snRNPs recycled
Describe ribozymes
a sequence of RNA that has catalytic activity
Which snRNA is a ribozyme? Where is this snRNA located?
the snRNA in U6 is a ribozyme
What is the catalytic activity of the snRNA in U6?
endonuclease activity that cleaves the intron
Why do non-coding RNAs have the snRNA in U6?
non coding RNAs are usually folded so the endonuclease activity helps produce their catalytic activity
What signifies a successful splice event?
an exon junction complex = 2 adjacent exons
What does the snRNA have complementarity with?
the snRNA in the snRNP complex has complementarity with the DNA
In relation to transcription, when are the introns cleaved?
as the pre mRNA leaves RNAPII (before the transcript is finished)
T or F: some spliceosome components are attached to the CTD
true
What is the purpose of splicing?
it introduces plasticity into the transcription process which increases diversity in the transcripts
What are 3 ways splicing introduces diversity in transcripts?
- individual splice sites can be skipped
- exons can be removed
- the first exon and last exon can vary
What happens if individual splice sites are skipped?
some introns are not cleaved
How many alternative mRNA sequences can potentially be made by alternative splicing?
hundreds from a single gene
What is the function of alternative splicing?
to increase protein complexity without increasing the number of genes
T or F: alternative splicing increases protein complexity by increasing the number of genes
False
it does not increase the number of genes
T or F: alternative splicing does not occur in very many human genes
False! alternative splicing happens in almost all human genes
In eukaryotes, where is the completed mRNA transported FROM and TO?
from the nucleus
through the nuclear pores
to the cytosol
Does mRNA move passively through the nuclear pores? why/why not?
No, it is complexed with proteins so it cannot move freely through the pores
What does mRNA require to move through the nuclear pores? why?
an exportin protein because it is attached to other proteins and cannot diffuse freely
What happens to mRNA in the cytosol?
eventually it is degraded
What happens to the nucleotides in mRNA after it is degraded in the cytosol?
they are brought back to the nucleus to be recycled
What 4 things are bound to a mature mRNA that is ready to leave the nucleus?
many non-snRNP proteins
a cap-binding protein
poly-A-binding proteins
exportin
exon junction complex
What happens to the proteins bound to the mRNA when it enters the cytosol?
some proteins leave the mRNA (ex. exportin) and some stay bound
more proteins involved in translation bind in the cytosol
What effect do the proteins that remain bound to the mRNA in the cytosol have on the mRNA?
they can effect the efficiency of translation
they can effect the destination of the transcript
