RNA metabolism and processing Flashcards
How does RNA differ from DNA?
- RNAs have OH group on 2nd carbon of sugar (oxy ribonuclease) - DNA is deoxy- doesn’t have O on 2’C
- RNA is single stranded
- Uracil is not found in DNA
- RNA has both genetic and catalytic functions; DNA only has genetic function
What are the molecules that can act as informational transmitter and catalyst?
RNA only
What is transcriptome?
Collection of all transcripts in a cell
What are similarities between RNA and DNA synthesis?
v Addition of complementary nucleotide
v Requirement of template
v Direction of synthesis 5’->3’
What are differences between RNA and DNA synthesis ?
v No primer required: DNA synthesis requires a primer, RNA synthesis doesn’t
v Segment of DNA is used as template: Whole RNA is used as a template
v One template strand: 1 in RNA, 2 strands in DNA
What is template strand?
Any strands used by polymerase is called a template strand
What is a transcript?
Any sequence made by polymerase is called transcript
In which direction does RNA pol II read the transcript?
3’ to 5’
Which strand is coding strand almost identical to?
To the RNA transcript strand
the only difference from non template strand is that transcript strand has U instead of T
What is the width between the 2 nucleotides? What is it created by?
1.08 nm - hydrogen bond
What is the rule of base pairing
Purine + pyrimidine
G or A + C or T or U
In which form do all nucleotides come in?
As triphosphates
How does pol 2 attach nucleotides?
nucleotides come in as triphosphates
Enzyme attacks alpha phosphate and throws away gamma and betta
This phosphate groups are at 5th carbon
Remaining alpha group interacts with 3rd carbon of existing ribose
5’C 3’C bond is formed - phosphodiester
What does pol II require?
DNA template, all 4 ribo-NTDs, Mg2+
On which strand can a coding strand be located on?
On either strand
What is TSS?
Transcription start site
What is found downstream of TSS
Gene sequence
What is found upstream of TSS
Upstream of TSS is identified as promoter as this is where all the regulatory elements are present. This is also where Polymerase binds
What are consequence sequences?
Consequence sequences are similarly looking sequences. These are typical elements one can see in eukaryotic genes
RNA polymerase binds to specific sequences in the DNA called ___
RNA polymerase binds to specific sequences in the DNA called promoters
What is the other name for transcription cycle in prokaryotes?
Sigma cycle
Describe initiation of sigma cycle
Has to start with initial transcription factors that help Pol to come and bind
Initial TF that comes in is called a sigma factor that binds to promoter
This binding brings in RNA Pol
Once sigma and RNA pol bind, they initiate transcription process
Describe elongation of sigma cycle
Pol can read DNA sequence and assemble nucleotides that are complementary to it
As transcription begins Pol leaves the promoter and goes forward
Sigma factors is not needed any more A- it leaves
NusA comes in to help pol move forward
What is the function of sigma?
Bind pol in prokaryotes
What is NusA
It is a protein that helps the pol to move down (helps with elongation) the gene and make the transcript
Describe termination of sigma cycle
Transcription is terminated
NusA dissociates and the RNA polymerase is recycled
What are the 3 types of polymerases?
Pol I: synthesis of rRNA
Pol II: synthesis of mRNA and specialized RNA E.g. microRNA lncRNA
Pol III: synthesis of tRNA, 5s rRNA and specialized RNA
How many polymerases do prokaryotes have?`
1
Where does transcribed mRNA exit the polymerase?
At a cleft
How many subunits does RNA pol ii have?
12
What are the 4 steps of making mRNA?
Assembly
Initiation
Elongation
Termination
Describe pol ii assembly
- TATA binding protein binds (TBP) to TATA box TBP is bound by transcription factor TFIIB
- TFIIB forms pre-initiation complex when POL II binds to TFIIB. 10-12 basal TF are involved.
- Helicase activity promotes the unwinding of DNA near the RNA start site- around 17 nucleotides are open
This opening of DNA strand is called transcription bubble result in an open initiation complex
As pol moves this bubble is closed up
Describe pol ii initiation
RNA pol has to move forward to start initiation
This movement require some modification which happens in CTD domain (carboxyterminal domain ) which is identified by a little tail
For any protein there’s carboxy terminal
CTD has to be phosphorylated for pol II to move forward- done by TF2H
With phosphorylation of CTD pol II escapes the promoter- transcription is initiated
What does ctd contain?
v Contains heptad AA repeats
v About 52 repeats in humans
Describe pol ii elongation
TF2H and other basal TF are no longer needed, so they exit the system
Elongation factors are needed to keep moving forward so they come in and assist pol II
Describe pol ii termination
Elongation factors leave. CTD is dephosphorylated
Termination factors come in to help pol2 to stop transcribing
Transcription stops, machinery is disassembled. Another transcription factor can happen again
What is the function of TFIIA?
Stabilizes binding of TFIIB and TBP to the promoter
What is the function of TFIIB?
Binds to TBP; recruits Pol II–TFIIF complex
What are the 3 mRNA modifications?
- 5 cap addition
- Splicing
Poly(A)tail
- Splicing
When does 5’ capping occur?
Early- after 20 30 base pairs
What is the purpose of 5’ capping?
The 5 cap helps protect mRNA from ribonucleases which cleave RNA
What is 5’ cap
It is a residue of 7- methyl guanosine linked to the 5-terminal residue of the mRNA through an unusual 5,5-triphosphate linkage
What is the linkage in 5’ cap?
unusual 5,5-triphosphate linkage
Steps of 5” cap
- Phosphohydrolase removes 1 phosphate group from 5’ of mRNA - from the first phosphate- making it diphosphate
2.Guanylyl transferase brings in GTP (Guanine triphosphate), removes 2 phosphates and the rest is added to the 5’ end of mRNA
Now there are 3 phosphates with 5’ to 5’ bonding - The guanine is subsequently methylated at N-7 byGuanine-7-methyltransferase
- All three of the capping enzymes, and the 5’ end of the transcript itself, are associated with the RNA polymerase II CTD until the cap is synthesized. The capped 5’ end is then released from the capping enzymes and
bound by the cap-binding complex
The attachment is carried out by 3 enzymes
Why is CTD important in 5’ cap?
Cap synthesizing complex, which contains enzymes needed for 5’ capping, comes in and bind to CTD, this results in 5’ capping
How do CSC, CTD and CBC interact?
CSC (Cap synthesizing complex) contains enzymes needed for 5’ capping, comes in and bind to CTD
CBC (cap binding complex) keeps cap bound to CTD
Where are introns not found?
Histones - RNA splicing cannot occur there
How long/short are introns and exons are?
Exons are short (<1000 bp); Introns are long (up to 20K bp)
In which groups of introns can self-splicing occur?
Group 1 and 2 e.g. mitochondrial genes in mitochondria
Describe self-splicing
Requires a guanine nucleoside or nucleotide cofactor.
5’ end of intron has UA sequence
3’ end of intron has GU sequence
The 3’ OH of guanosine (GTP) acts as a nucleophile, attacking the phosphate at the 5’ splice site breaking phosphodiester bond between UA
The guanosine 3-hydroxyl group forms a normal 3,5-phosphodiester bond with the 5’ end of the intron
The 3’ OH of the 5’ exon becomes the nucleophile, completing the reaction.
UU bond
What is a spliceosome and what is is made of?
large protein complex
Made up of specialized RNA-protein complexes (has both RNA and protein parts), Small Nuclear RiboNucleoProteins- snRNPs- small nuclear RNAs
Describe snRNPs
small nuclear ribonucleoproteins Each snRNPs contains one of a class of eukaryotic RNAs known as small nuclear RNAs (snRNAs).
What are the 5 snRNAs? Where are they found
Five snRNAs (U1, U2, U4, U5, and U6) involved in splicing reactions are generally found in abundance in eukaryotic nuclei.
In which groups of introns can splicing with spliceosome occur?
Groups 3 and 4
Group 3 present in most mRNA
Group 4 are present in most tRNAs
Describe splicing with spliceosome
Introns that has to be spliced has 2 ends:
3’ end- donor site, 5’ end acceptor site
Also have a branching site
U1 and U2 snRNAs come in - Binding requires energy
U1 goes to 5’, end U2 goes to branching sequence where there’s an A
U5 comes in and binds to 3’ end
Addition U4 and U6 creates a spliceosome
GU of the donor site and A of branching sequence are brought together to bond and form a lariat
This frees up 3’OH of the exon which can attack 3’ end of an intron which cleaves the 3’ to 5’ bond completing splicing
2 exons are brought in together
3 structures of Nucleotides sequences in introns that are spliced by Spliceosome:
- Donor site: 5’ end of intron has dinucleotide G-U
- Acceptor site: 3’ end has A-G dinucleotide (2 nucleotides)
Branching site: Upstream of 5’ end there is sequence that is identified by A
- Acceptor site: 3’ end has A-G dinucleotide (2 nucleotides)
Where do snRNPs bind?
Close to CTD
What is a poly A tail? What is it’s purpose?
Poly (A) tail -a string of 80 to 250 A residues at 3’ end of mRNA in most eukaryotes
help protect mRNA from enzymatic destruction; serves as a binding site for one ore more sepcific proteins
What is a cleavage signal?
it is is a sequence made of As and Us - typically involves AAUAAA
Describe how poly a tail is made
Adaptor proteins and enzymes come together and recognize this cleavage sequence where polyadenylate polymerase and endonuclease form a complex
When this complex is assembled cleavage occurs almost right after this sequence by endonuclease
Cleaves out final portion of RNA that is transcribed
Polyadenylate polymerase adds a several As (can be hundreds) to the end, forming a poly-A tail
What happens to introns that are retained in alternative splicing?
They become exons
What do RNAs with catalytic function use as cofactors?
Mainly metals
Define nontemplate strand
The DNA strand complementary to the template, the nontemplate strand, or coding strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA
Which enzymes carry out the synthesis of the cap?
It is carried out by enzymes attached to the CTD of Pol IIt
What is cap attached to after synthesis and how ?
It remains attached to the CTD through an association with the cap-binding complex (CBC)
Which group of introns requires ATP to be spliced by spliceosome?
Group 3 and 4 e.g tRNA
Where do components of spliceosome bind to at Pol II?
CTD
Give examples of genes that can do self splicing
Mitochondrial genes
Which type of splicing requires energy? Which doesn’t?
Self-splicing doesn’t require energy
Splicing by spliceosome does
