Lecture 32: RNA Synthesis

Question 1

4 types of RNA from DNA templates

Answer 1

mRNA
rRNA (ribosomal)
tRNA
small nuclear RNAs

Question 2

Bactreial RNA polymerases

Answer 2

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

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

Question 3

3 RNA polymerases in eukaryotes

Answer 3

Pol 1 (synth rRNA)
Pol 2 (synth mRNA)
Pol 3 (synth tRNA, rRNA)

Question 4

mRNA

Answer 4

directs messages and sequence of amino acids in protein

Question 5

rRNA

Answer 5

large, part of ribosome

catalytic RNA

Question 6

tRNA

Answer 6

link between mRNA and amino acid

Question 7

base pairing of what?

Answer 7

tRNA and mRNA

Question 8

snRNA

Answer 8

regulatory functions

Question 9

Transcription

Answer 9

DNA dependent RNA synth

aka Gene Expression

Question 10

difference between transcirption in eukaryotes vs prokaryotes

Answer 10

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

Question 11

RNA splicing is to…

Answer 11

remove non-coding sequences (introns)

Question 12

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

Answer 12

C
U
A

Question 13

if CODING strand DNA is G… RNA is…

Answer 13

G

Question 14

RNA polymerase in prokaryotes

Answer 14

need to accesss template starnd of DNA to guide RNA synth

DOES NOT require pre-existing primer

Question 15

directoion of RNA synth

Answer 15

5’ to 3’

Question 16

base pairing in bacteria

Answer 16

leads to temp DNA-RNA hybrid at TRANSCRIPTION bubble

Question 17

bacteria template strand

Answer 17

RNA will be complimentary to this strand

Question 18

bacteria coding strand

Answer 18

has same sequence of RNA being created

complementary to template

Question 19

Transcirption bubble…

Answer 19

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

Question 20

DNA hybrid is..

Answer 20

pretty short

becomes single stranded as it exits

Question 21

enzymology of RNA synth

Answer 21

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

Question 22

RNA polymerase proofreading?

Answer 22

not clasic proofreading activity

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

Question 23

importance of magnesium ions in RNA pol

Answer 23

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

Question 24

Transcription generates _____ mRNA molecules

Answer 24

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)

Question 25

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

Answer 25

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

Question 26

winding problem

Answer 26

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

Question 27

what relives torsional stress?

Answer 27

topoisomerase

Question 28

True or false? RNA polymerase transcribes only one DNA strand

Answer 28

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

Question 29

Where does RNA synth begin?

Answer 29

at promoter sequence

Question 30

promoter sequences in bacterial genes

Answer 30

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

Question 31

when does the sigma factor fall off?

Answer 31

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

Question 32

- 10 and -35 regions recognized by...

Answer 32

sigma factor | directs -35 and -10 regions to form initial closed complex

Question 33

Bacterial RNA pol: binding

Answer 33

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)

Question 34

DNA footprinting

Answer 34

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

Question 35

DNA footprinting... what happens?

Answer 35

you end up with fragments that are cut at different sites | you can see where the RNA pol binds by running on a gel

Question 36

3 conditions for footprinting

Answer 36

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

Question 37

DNA footprinting: Blank regions

Answer 37

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

Question 38

STUDY SLIDE 13

Answer 38

STUDY SLIDE 13

Question 39

template strand is... SEE SLIDE 14 TOO

Answer 39

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

Question 40

Eukarotic Polymerases: RNA pol 1

Answer 40

makes ribosomal RNA | promoters: INR sequence and UPE (upstream promoter element) element

Question 41

RNA Pol 2

Answer 41

makes messenger RNA | promoters: INR sequence and TATA box (or DPE sequence)

Question 42

RNA Pol 3

Answer 42

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

Question 43

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

Answer 43

recognition of promoters | also, the pols work differently

Question 44

alpha amanitin

Answer 44

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

Question 45

TATA binding Protein (TBP)

Answer 45

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

Question 46

RNA Pol 2 and promoter sequence

Answer 46

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

Question 47

Pol 2 in Yeast

Answer 47

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

Question 48

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

Answer 48

Sigma factor! both help to position polymerase at right place to start transcription not related at all structurally