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Bchem 393 > Gene Expression And RNA Processing > Flashcards

Gene Expression And RNA Processing Flashcards

1
Q

What is transcription in E. coli

What are the special things about it

A

DNA to RNA

RNA is made 5’ to 3’ and uses the 3-5 stand of dna to transcribe

Is slower than dna replication because it transcribed 50nucletides/sec

DNA does 1000/sec in replication

Doesn’t need primer to start transcription because of the ppp and the start of the rna strand

Doesn’t need helicase to split apart the dna strands

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2
Q

What is needed for transcription to happen

A

NTP (not dntp)
RNA polymerase
DNA template
Divalent cation like mg2+ (in rna pol active site)

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3
Q

What are the subunits of RNA pol in E. coli

A

Two alpha
Beta
Beta prime
Omega
Sigma 70 (70kda)

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4
Q

What do the 2 alpha subunits do

A

The assemble the core rna pol enzyme and interact with regulatory factors

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5
Q

What does the beta and beta prime subunits do

A

Catalysis of transcription

Interacts with dna and rna

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6
Q

What does the omega subunit do

A

Restore the denature polymerase to its native form

It’s important for the structure of the enzyme

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7
Q

What does the sigma 70 subunit do

A

Takes part in promoter recognition

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8
Q

What subunits form the rna pol core enzymesm

What about its active site

A

Aplha x2
Beta
Beta prime
Omega

Beta and beta prime

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9
Q

What is a holoenzyme

A

The core enzyme and the sigma subunit

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10
Q

The active site of rna pol is

A

In between beta and beta prime

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11
Q

Where is transcription initiated

A

The promoter

Has the -10 (also called pribnow box) and -35 sequences upstream of the +1 start site of the coding strand

These sequences are 6 bp long

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12
Q

How is transcription initiated

A

First the sigma subunit of the RNA pol decreases rna pols affinity for dna

This lets the rna pol move quickly along the dna template strand to find the promoter

The sigma subunit then recognizes the promoter (-35 and -10)

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13
Q

What happens to the sigma subunit after it’s found the promoter and many rna nucleotides have been made?

A

The sigma subunit falls of the core of the holoenzyme

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14
Q

Does rna synthesis need a primer to start

A

No

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15
Q

How is elongation carried out in rna transcription

Where does the already made 5 prime rna go

A

After forming the first phosphodiester linkage, elongation happens

New ntps are added to the growing strand at the 3’ end

The 5’ already made rna is released though a channel in the enzyme

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16
Q

What is the transcription bubble
How long is it

A

The denatruated area of the dna that’s unwounds and rna is being made

17bp long

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17
Q

Is the reaction of pyrophosphate (PPi) to 2Pi exergonic or endergonic

A

Exergonic so the reaction of adding NTPs is driven forward

18
Q

What types of termination are the in rna transcription

A

Intrinsic termination

Protien dependent termination

19
Q

How does intrinsic termination of transcription happen

A

The rna product froms a hair pin because of complimentary pairs of G AND C that are repeation in the dna

This makes the rna pol pause

A sequence of a bunch of U is after this hairpin in the rna

These A to U bp bonds (rU-dA) are weak because there’s only 2 h bond between them

So the rna strand dissociates from the template dna strand and the RNA pol enzyme

20
Q

What is protien dependent termination

A

This is when the rna pol need help in stopping where there isn’t a hairpin and UUUU repeat

A protien called rho which is a helicase (hexameric and hydrolyzes atp)

The rho protien pulls the rna strand away from the DNA through its centre.

21
Q

What is different in transcription between prokaryotes and eukaryotes

A

Prokaryotes: transcription happens at the same time as translation (The ribosome binds right as the rna pol makes a new strand)

More simple control elements (less protiens used)

The termination signal is the GC hair pin

The mRNA transcript isnt processed and transported across a membrane

Eukaryotes: transcription happens in the nucleus, the rna transcript is processed, then sent to the cytoplasm

Translation happens in the cytoplasm

Termination signal is the Poly A tail

22
Q

What are all of the promoter elements for rna pol

There are 6

A

They define the start site of transcription on the dna and recruit the rna pol

Cis acting elements

TATA box

Inr (initiator element)

DPE (downstream core promoter element)

Enhancer

CAAT box and GC box

23
Q

What are cis acting elements

A

DNA sequences that regulate expression of a gene that’s on the same molecule of dna

24
Q

What is the tata box and the Inr

A

Together they make up the promoter

TATA box is -25 bp upstream of the Inr

The Inr is the initiator element and it at the +1 site of the dna

25
Q

What is the DPE

A

The downstream promoter element which is +30 bp down stream from the inr (+1)

It can also make up the promoter with Inr

26
Q

What is the enhancer

A

Can be more than one kb upstream from the start site

27
Q

What is the CAAT box and the GC box

A

They are involved in regulation and initiation of transcription

They’re involved in transcription of genes that that are constitutively (always) expressed

They’re at the -40 to-150 region upstream of the Inr

28
Q

What is the pre initiation complex (PIC)

How does it start

A

This is formed when transcription factors bind cos acting element that recruit RNA pol 11

This complex is first started by the TATA binding protein (TBP) on TF11D recognizing the TATA box and binding to it

TF11D is a dynamic protien meaning it has a lobe (lobe A) which moves as the TBP is looking for the TATA box

29
Q

How is the PIC formed

A

First the TBP on TF11D recognized and binds to the TATA box on the dna after moving through many diff confirmations

TF11A binds to TF11D and stabilizes the interactions with the promoter region

TBP engages the promoter by bending the DNA sequnce

TF11B binds and recruited RNA POL 11 , TF11F and E

The. The last step

30
Q

What is the last step of the PIC forming

A

The TF11H helicase bind and unwinds the dna

It the phosphorylates the c terminal domain of RNA pol 11

This phosphorylation causes a transition from initiation to elongation

and recruitment of rna processing enzymes

31
Q

Do prokaryotes need a helicase to unwind the dna For rna transcription

A

No, the rna pol does it itself

32
Q

In what way does the PIC regulate transcription

How can transcription be more regulated

A

At low levels, It give low level of gene expression

More transcription factors that bind to other sites on the dna can stimulate higher or lower levels of transcription

The gene as expressed in a tissue specific manner

33
Q

What is combinatorial control

A

When one transcription factor on the dna has little influence on its expression

So many other transcription factors are needed to make a complex that either stimulates or repressed transcription

34
Q

How is mRNA processed in eukaryotes

A

A 5’ cap and a poly a tail is added to the rna strand

Through splicing where The non coding region (introns) are removed and the exons (coding) are joined together

Splicing can make many different combos of genes based on how the mRNA is spliced

35
Q

How is the 5’ cap added to the 5’ end of men’s

A

The 5’ triphopsate of the rna has one phosphoryl group removed by phosphatase

Then the 5’ dihposphate end attacks the alpha phosphorous of GTP (guanalyl transferase does this)

The N7 of the guanine from GTP gets a methyl group on it, and the 2’oh of the divided nest the guanine also get methylated (by guanylmethyltransferase)

36
Q

Why does mRNA have a 5’ cap

A

Do protect the 5’ end of rna from nucleases and enhance translation

37
Q

How is the poly A tail processed

A

The CPSF (cleavage and polyadenylation specificity factor) recognizes the cleavage signal of the rna

Then an endonuclease cleaves the part of the rna with the signal

Then poly A polymerase adds a 250 bp adenylate (Adenines) to the cleaved 3’ end

38
Q

What does the poly a tail help with

A

Stability and translation

39
Q

How does splicing occur

A

The spliceosome which is a complex of rna / protien recognizes conserved (specific) sequences in the introns

These sequences are right next to the exons, so they are split right next to the exons

Then the spliceosome joins together the exons

40
Q

Describe the process of splicing up to the lariats intermediate

A

The OH of the introns adenylate breaks the ester bond between the intron and the exon at the introns 5’ splice site

(1transwsterification reaction)

This forms a lariat intermediate where the two introns form a phosphodiester bond

The 3’ exon is still attached to the intro

41
Q

What happens in splicing after the l lariat intermediate is formed

A

The released 5’ exon has a free 3’ oh group that attacks the ester bond of the second splice site

(2nd transesterification reaction)

This joins the two exons together and releases the intron in the lariat form goth a free 3’ oh

42
Q

What is a transesterification reaction and how many does the spliceosome do

A

A reaction between and oh and and ester to make a new oh and ester

2 reactions

Bchem 393 (31 decks)

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