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RNA metabolism and processing Flashcards

1
Q

How does RNA differ from DNA?

A
  • 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
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2
Q

What are the molecules that can act as informational transmitter and catalyst?

A

RNA only

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3
Q

What is transcriptome?

A

Collection of all transcripts in a cell

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4
Q

What are similarities between RNA and DNA synthesis?

A

v Addition of complementary nucleotide
v Requirement of template
v Direction of synthesis 5’->3’

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5
Q

What are differences between RNA and DNA synthesis ?

A

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

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6
Q

What is template strand?

A

Any strands used by polymerase is called a template strand

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7
Q

What is a transcript?

A

Any sequence made by polymerase is called transcript

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8
Q

In which direction does RNA pol II read the transcript?

A

3’ to 5’

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9
Q

Which strand is coding strand almost identical to?

A

To the RNA transcript strand

the only difference from non template strand is that transcript strand has U instead of T

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10
Q

What is the width between the 2 nucleotides? What is it created by?

A

1.08 nm - hydrogen bond

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11
Q

What is the rule of base pairing

A

Purine + pyrimidine

G or A + C or T or U

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12
Q

In which form do all nucleotides come in?

A

As triphosphates

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13
Q

How does pol 2 attach nucleotides?

A

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

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14
Q

What does pol II require?

A

DNA template, all 4 ribo-NTDs, Mg2+

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15
Q

On which strand can a coding strand be located on?

A

On either strand

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16
Q

What is TSS?

A

Transcription start site

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17
Q

What is found downstream of TSS

A

Gene sequence

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18
Q

What is found upstream of TSS

A

Upstream of TSS is identified as promoter as this is where all the regulatory elements are present. This is also where Polymerase binds

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19
Q

What are consequence sequences?

A

Consequence sequences are similarly looking sequences. These are typical elements one can see in eukaryotic genes

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20
Q

RNA polymerase binds to specific sequences in the DNA called ___

A

RNA polymerase binds to specific sequences in the DNA called promoters

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21
Q

What is the other name for transcription cycle in prokaryotes?

A

Sigma cycle

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22
Q

Describe initiation of sigma cycle

A

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

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23
Q

Describe elongation of sigma cycle

A

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

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24
Q

What is the function of sigma?

A

Bind pol in prokaryotes

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25
What is NusA
It is a protein that helps the pol to move down (helps with elongation) the gene and make the transcript
26
Describe termination of sigma cycle
Transcription is terminated | NusA dissociates and the RNA polymerase is recycled
27
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
28
How many polymerases do prokaryotes have?`
1
29
Where does transcribed mRNA exit the polymerase?
At a cleft
30
How many subunits does RNA pol ii have?
12
31
What are the 4 steps of making mRNA?
Assembly Initiation Elongation Termination
32
Describe pol ii assembly
1. TATA binding protein binds (TBP) to TATA box TBP is bound by transcription factor TFIIB 2. TFIIB forms pre-initiation complex when POL II binds to TFIIB. 10-12 basal TF are involved. 3. 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
33
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
34
What does ctd contain?
v Contains heptad AA repeats | v About 52 repeats in humans
35
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
36
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
37
What is the function of TFIIA?
Stabilizes binding of TFIIB and TBP to the promoter
38
What is the function of TFIIB?
Binds to TBP; recruits Pol II–TFIIF complex
39
What are the 3 mRNA modifications?
- 5 cap addition - Splicing Poly(A)tail
40
When does 5' capping occur?
Early- after 20 30 base pairs
41
What is the purpose of 5' capping?
The 5 cap helps protect mRNA from ribonucleases which cleave RNA
42
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
43
What is the linkage in 5' cap?
unusual 5,5-triphosphate linkage
44
Steps of 5" cap
1. 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 2. The guanine is subsequently methylated at N-7 byGuanine-7-methyltransferase 3. 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
45
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
46
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
47
Where are introns not found?
Histones - RNA splicing cannot occur there
48
How long/short are introns and exons are?
Exons are short (<1000 bp); Introns are long (up to 20K bp)
49
In which groups of introns can self-splicing occur?
Group 1 and 2 e.g. mitochondrial genes in mitochondria
50
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
51
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
52
Describe snRNPs
``` small nuclear ribonucleoproteins Each snRNPs contains one of a class of eukaryotic RNAs known as small nuclear RNAs (snRNAs). ```
53
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.
54
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
55
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
56
3 structures of Nucleotides sequences in introns that are spliced by Spliceosome:
1. Donor site: 5' end of intron has dinucleotide G-U 2. Acceptor site: 3' end has A-G dinucleotide (2 nucleotides) Branching site: Upstream of 5' end there is sequence that is identified by A
57
Where do snRNPs bind?
Close to CTD
58
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
59
What is a cleavage signal?
it is is a sequence made of As and Us - typically involves AAUAAA
60
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
61
What happens to introns that are retained in alternative splicing?
They become exons
62
What do RNAs with catalytic function use as cofactors?
Mainly metals
63
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
64
Which enzymes carry out the synthesis of the cap?
It is carried out by enzymes attached to the CTD of Pol IIt
65
What is cap attached to after synthesis and how ?
It remains attached to the CTD through an association with the cap-binding complex (CBC)
66
Which group of introns requires ATP to be spliced by spliceosome?
Group 3 and 4 e.g tRNA
67
Where do components of spliceosome bind to at Pol II?
CTD
68
Give examples of genes that can do self splicing
Mitochondrial genes
69
Which type of splicing requires energy? Which doesn't?
Self-splicing doesn't require energy | Splicing by spliceosome does

BC 2- (19 decks)

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