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Biochem 385 Unit 3 > Lecture 32: RNA Synthesis > Flashcards

Lecture 32: RNA Synthesis Flashcards

1
Q

4 types of RNA from DNA templates

A

mRNA
rRNA (ribosomal)
tRNA
small nuclear RNAs

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2
Q

Bactreial RNA polymerases

A

bind to specific DNA Promoter Sequences (on 5’ end)

initiatin of synth starts with open complex formation and copy the template strand

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3
Q

3 RNA polymerases in eukaryotes

A 
Pol 1 (synth rRNA)
Pol 2 (synth mRNA)
Pol 3 (synth tRNA, rRNA)
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4
Q

mRNA

A

directs messages and sequence of amino acids in protein

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5
Q

rRNA

A

large, part of ribosome

catalytic RNA

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6
Q

tRNA

A

link between mRNA and amino acid

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7
Q

base pairing of what?

A

tRNA and mRNA

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8
Q

snRNA

A

regulatory functions

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9
Q

Transcription

A

DNA dependent RNA synth

aka Gene Expression

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10
Q

difference between transcirption in eukaryotes vs prokaryotes

A

Eu: transcribe DNA in nucleus, translate mRNA in cytoplasm. processesd by RNA splicing
Pro: transcription and translation at same time

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11
Q

RNA splicing is to…

A

remove non-coding sequences (introns)

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12
Q

if templates strand DNA is… then RNA is…
G
A
T

A

C
U
A

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13
Q

if CODING strand DNA is G… RNA is…

A

G

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14
Q

RNA polymerase in prokaryotes

A

need to accesss template starnd of DNA to guide RNA synth

DOES NOT require pre-existing primer

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15
Q

directoion of RNA synth

A

5’ to 3’

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16
Q

base pairing in bacteria

A

leads to temp DNA-RNA hybrid at TRANSCRIPTION bubble

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17
Q

bacteria template strand

A

RNA will be complimentary to this strand

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18
Q

bacteria coding strand

A

has same sequence of RNA being created

complementary to template

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19
Q

Transcirption bubble…

A 
moves 
base pairs
reseals
as complex moves
opens so that complementary pairing can occur
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20
Q

DNA hybrid is..

A

pretty short

becomes single stranded as it exits

21
Q

enzymology of RNA synth

A

like DNA synth
nucleophilic attack on 3’ OH group on alpha phosphate
generation of new phosphodiester bond with basepairing

22
Q

RNA polymerase proofreading?

A

not clasic proofreading activity

but does “back-up” and resynthesize strand when mismatch is made

23
Q

importance of magnesium ions in RNA pol

A

positive charges interact with negative charges of phosphate
stabilization
positioned by aspartate residues

24
Q

Transcription generates _____ mRNA molecules

A

MANY!
each gene produces 1000 to 100000 transcripts
error rate is about 1 error per 10,000 nucleotides
(VERY similar to DNA polymerization error rate)

25
Q

RNA pol does not have proofreading activity, and there is no RNA repair mechanism. Why is an error rate of 10^-4 nucleotides acceptable?

A

errors won’t persist because proteins made will only cause a temporary problem
odds of one being bad is lower???

26
Q

winding problem

A

as transcription bubble moves along the DNA double helix, it creates stress and induces super coils on both sides of bubble
causes Torsional stress

27
Q

what relives torsional stress?

A

topoisomerase

28
Q

True or false? RNA polymerase transcribes only one DNA strand

A

FALSE
it transcribes both
CODING AND TEMPLATE STRANDS AREN’T ABSOLUTE
regions may differ
there is not much overlap so that proteins aren’t coded in both template and complementary strand

29
Q

Where does RNA synth begin?

A

at promoter sequence

30
Q

promoter sequences in bacterial genes

A

evolutionary conserved DNA sequences that are
-35 and -10 regions relative to +1 RNA start site
spacers between these 3 regions

31
Q

when does the sigma factor fall off?

A

when the polymerase moves away from the promoter sequence (the minus regions)

32
Q
  • 10 and -35 regions recognized by…
A

sigma factor

directs -35 and -10 regions to form initial closed complex

33
Q

Bacterial RNA pol: binding

A

RNA pol binds to promoter region as complex containing sigma factor
closed complex formed
complex binding leads to open complex
transcription starts and sigma factor falls off (sigma used again)

34
Q

DNA footprinting

A

to find where RNA pol binds to DNA sequence
lable the DNAs, split into 2 grouos, 1 w/ protein of interest added, one w/0
add DNAse1 (enough to cut DNA once)
then we end up with DNA cuts of different lenghts
get cuts at every position
IF PROTEIN IS PRESENT, cutting can’t happen b/c DNA is protected by binding of RNA Pol
DNA suspected radilabled, incubate w/ or w/o RNA pol

35
Q

DNA footprinting… what happens?

A

you end up with fragments that are cut at different sites

you can see where the RNA pol binds by running on a gel

36
Q

3 conditions for footprinting

A

1) DNA must be labled on only 1 end of fragment (reference point)
2) DNase 1 digestion must be incomplete to generate the different fragment lengths
3) RNA pol must bind specifically to single sequences w/in fragment

37
Q

DNA footprinting: Blank regions

A

is where it is bound by RNA pol, so protected from cleavage (bands only show up where there was cleavage)
(if we had no protein, cutting could happen anywhere)
blank regions are where “footprinting” is occuring

38
Q

STUDY SLIDE 13

A

STUDY SLIDE 13

39
Q

template strand is…

SEE SLIDE 14 TOO

A

complementary to growing RNA strand
so T would encode A, A would encode U… etc.

the complimentary coding strand is the same as the RNA strand

40
Q

Eukarotic Polymerases: RNA pol 1

A

makes ribosomal RNA

promoters: INR sequence and UPE (upstream promoter element) element

41
Q

RNA Pol 2

A

makes messenger RNA

promoters: INR sequence and TATA box (or DPE sequence)

42
Q

RNA Pol 3

A

makes tRNA and one of rRNAs
promoters: blocks
A block and C block (for rRNA)
A block and B block (for tRNA)

43
Q

how does an RNA pol know which RNA it will be making?

A

recognition of promoters

also, the pols work differently

44
Q

alpha amanitin

A 
inhibits Pol 2
high concs inhibit Pol 3
doesn't inhibit Pol 1
Why is this important?
used to find differences between the pols
45
Q

TATA binding Protein (TBP)

A

directs RNA Pol II compex to a T-A-T-A sequence in the promoter region of protein coding genes

TATA sequence positions the RNA Pol II complex w/in the gene promoter

DNA piece is really bent. TATA is easier to bend and open up, makes it easier to form open complex

46
Q

RNA Pol 2 and promoter sequence

A

needs to recognize promoter sequence
binds to TATA and D. Add factors and RNA Pol 2. HUGE complex
once complex is made at promoter, transcription can start
then a bunch of factors fall off. D, TATA, 2F, and Pol 2 are all thats left

47
Q

Pol 2 in Yeast

A

has 12 subunits
RNA pol complex unwinds DNA helix
polymerizes nascent mRNA transcript
lots of other stuff happens too

48
Q

What is the bacterial equivalent of the TATA binding protein (TBP)

A

Sigma factor!
both help to position polymerase at right place to start transcription

not related at all structurally

Biochem 385 Unit 3 (11 decks)

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