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BC 2- > RNA metabolism and processing > Flashcards

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
Q

What is NusA

A

It is a protein that helps the pol to move down (helps with elongation) the gene and make the transcript

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26
Q

Describe termination of sigma cycle

A

Transcription is terminated

NusA dissociates and the RNA polymerase is recycled

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27
Q

What are the 3 types of polymerases?

A

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

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28
Q

How many polymerases do prokaryotes have?`

A

1

29
Q

Where does transcribed mRNA exit the polymerase?

A

At a cleft

30
Q

How many subunits does RNA pol ii have?

A

12

31
Q

What are the 4 steps of making mRNA?

A

Assembly
Initiation
Elongation
Termination

32
Q

Describe pol ii assembly

A
  1. TATA binding protein binds (TBP) to TATA box TBP is bound by transcription factor TFIIB
    1. TFIIB forms pre-initiation complex when POL II binds to TFIIB. 10-12 basal TF are involved.
    2. 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
Q

Describe pol ii initiation

A

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
Q

What does ctd contain?

A

v Contains heptad AA repeats

v About 52 repeats in humans

35
Q

Describe pol ii elongation

A

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
Q

Describe pol ii termination

A

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
Q

What is the function of TFIIA?

A

Stabilizes binding of TFIIB and TBP to the promoter

38
Q

What is the function of TFIIB?

A

Binds to TBP; recruits Pol II–TFIIF complex

39
Q

What are the 3 mRNA modifications?

A
  • 5 cap addition
    • Splicing
      Poly(A)tail
40
Q

When does 5’ capping occur?

A

Early- after 20 30 base pairs

41
Q

What is the purpose of 5’ capping?

A

The 5 cap helps protect mRNA from ribonucleases which cleave RNA

42
Q

What is 5’ cap

A

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
Q

What is the linkage in 5’ cap?

A

unusual 5,5-triphosphate linkage

44
Q

Steps of 5” cap

A
  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
Q

Why is CTD important in 5’ cap?

A

Cap synthesizing complex, which contains enzymes needed for 5’ capping, comes in and bind to CTD, this results in 5’ capping

46
Q

How do CSC, CTD and CBC interact?

A

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
Q

Where are introns not found?

A

Histones - RNA splicing cannot occur there

48
Q

How long/short are introns and exons are?

A

Exons are short (<1000 bp); Introns are long (up to 20K bp)

49
Q

In which groups of introns can self-splicing occur?

A

Group 1 and 2 e.g. mitochondrial genes in mitochondria

50
Q

Describe self-splicing

A

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
Q

What is a spliceosome and what is is made of?

A

large protein complex
Made up of specialized RNA-protein complexes (has both RNA and protein parts), Small Nuclear RiboNucleoProteins- snRNPs- small nuclear RNAs

52
Q

Describe snRNPs

A 
small nuclear ribonucleoproteins
Each snRNPs contains one of a class of eukaryotic RNAs known as small nuclear RNAs (snRNAs).
53
Q

What are the 5 snRNAs? Where are they found

A

Five snRNAs (U1, U2, U4, U5, and U6) involved in splicing reactions are generally found in abundance in eukaryotic nuclei.

54
Q

In which groups of introns can splicing with spliceosome occur?

A

Groups 3 and 4
Group 3 present in most mRNA
Group 4 are present in most tRNAs

55
Q

Describe splicing with spliceosome

A

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
Q

3 structures of Nucleotides sequences in introns that are spliced by Spliceosome:

A
  1. Donor site: 5’ end of intron has dinucleotide G-U
    1. 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
Q

Where do snRNPs bind?

A

Close to CTD

58
Q

What is a poly A tail? What is it’s purpose?

A

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
Q

What is a cleavage signal?

A

it is is a sequence made of As and Us - typically involves AAUAAA

60
Q

Describe how poly a tail is made

A

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
Q

What happens to introns that are retained in alternative splicing?

A

They become exons

62
Q

What do RNAs with catalytic function use as cofactors?

A

Mainly metals

63
Q

Define nontemplate strand

A

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
Q

Which enzymes carry out the synthesis of the cap?

A

It is carried out by enzymes attached to the CTD of Pol IIt

65
Q

What is cap attached to after synthesis and how ?

A

It remains attached to the CTD through an association with the cap-binding complex (CBC)

66
Q

Which group of introns requires ATP to be spliced by spliceosome?

A

Group 3 and 4 e.g tRNA

67
Q

Where do components of spliceosome bind to at Pol II?

A

CTD

68
Q

Give examples of genes that can do self splicing

A

Mitochondrial genes

69
Q

Which type of splicing requires energy? Which doesn’t?

A

Self-splicing doesn’t require energy

Splicing by spliceosome does

BC 2- (19 decks)

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