7. Gene expression

Question 1

what is the enzyme that carries out transcription

Answer 1

DNA- dependent RNA polymerase

• multisubunit complexes
• couples ribonucleoside triphosphates (driven by hydrolysis of PPi)

Question 2

Difference between polymerase in bacteria and eukaryotes

Answer 2

bacteria contain one enzyme that makes most of the RNA and eukaryotees contain 4 or 5

Question 3

describe prokaryotic RNA polymerase

Answer 3

• holoenzyme: sigma subunit enables RNA polymerase to recognise promoter regions in DNA

Question 4

holoenzyme

Answer 4

sigma subunit and core enzyme

Question 5

Answer 5

Question 6

name of template DNA

Answer 6

antisense/ non coding strand

Question 7

DNA sequence that has the same sequence as RNA

Answer 7

sense/ coding strand

Question 8

protein coding genes in eukaryotes and prokaryotes

Answer 8

structural genes

eukaryotes: transcribed individually
prokaryotes: tandem layout and transcribed together- operons giving rise to polycistronic mRNA

Question 9

How does RNAP recognise the correct DNA strand and initiate at the beginnning of a gene?

Answer 9

Binds to base sequence promoters

in bacteria promoter regon is reocognised by sigma factor (adaptor)

Question 10

What are promoters

Answer 10

40 bp sequence on 5’ side of transcription start site upstream of RNA starting nucleotide

written by sense strand so matches directionality of transcribed RNA

Question 11

What is the number assigned to the first base pair

Answer 11

+1 (no zero base)

Question 12

What is the pribnow box

Answer 12

a common sequence in the promoter region in prokaryotes

Question 13

Why can we identify promoters?

Answer 13

RNAP’s tight binding protects the sequence from being broken down by endonucleas DNAse I

mutations can effect RNAP binding

Question 14

what does RNAP holoenzyme binding lead to

Answer 14

DNA ‘melting’ (separation) in its vicinity

• transcription bubble

allows complementary RNA strand synth.

bubble travels with RNAP

Question 15

RNAP is processive- explain

Answer 15

• does not dissociate from template
• 1900 bp, up to 180 rotations
• lots of reactions until it leaves

Question 16

Describe the nature of transciption

feature of transcription in prokaryotes

Answer 16

rapid

RNA synth is initiated as often as sterically possible

protein synth can begin before RNA is completely synthesised

Question 17

Do eukaryote RNAP contain a sigma factor

Answer 17

no

acessory proteins ID promoter and recruit RNAPs

Question 18

Transcription termination in prokaryotes

Answer 18

• Contains series of 4-10 A•T bases
• A G + C-rich region with a palindromic sequence

The RNA transcript (‘intrinsic terminator’) forms a self-complementary “hairpin”

• causes RNAP to pause
• permits RNAP conformational change allowing termination

Question 19

What happens if E.coli do not display the intrinsic terminator

Answer 19

need the Rho factor

• a helicase that runs along RNA until encountering a paused RNAP
• Pushes RNAP, causes rewinding of dsDNA & release of RNA

Question 20

Eukaryotic RNAP

Answer 20

Question 21

eukaryotic vs prokaryotic RNAP

Answer 21

eukaryotic have a larger mass and greater subunit complexity

Question 22

RNAP II promoters are…

Answer 22

complex/ diverse

if they are selectively expressed they contain consensous sequences such as TATA box ( equivilent to Pribnow box)

25 bp upstreat from TSS

Question 23

sequences that encourage transcription

Answer 23

enhancers

recognised by transcription factors which can stimulate RNAP II binding and mediate selective gene expression in eukaryotes

Question 24

what can DNA bending do

Answer 24

cause an enhancer that is far from the promoter to interact with transcription-initiation complex

Question 25

General transcription factors

Answer 25

required for RNAP II transcription equivalent of σ factor other gene specific factors can enhance

Question 26

General transcription factors combine with enzyme and DNA to form

Answer 26

preinitiation complex

Question 27

where are GTF targeted to

Answer 27

TATA box by TATA binding protein

Question 28

what do eurkaryotes lack and what is done to overcome this

Answer 28

precise transcription termination sites transcript processing takes place later

Question 29

Inhibitors of transcription: RIfamycin

Answer 29

inhibit prokaryotic but not eukaryotic transcription, side effects Makes RNAPs stay in the promoter region, so proteins cannot be made until bacteria runs out of protein Prevent elongation (not promoter binding /initial bond formation) - Inactivated RNA polymerase remains bound - blocks further initiation

Question 30

Inhibitors of transcription: Actinomycin D

Answer 30

- Binds to duplex DNA (intercalates) RNAP and DNA polymerase cannot get past - Inhibits DNA replication & transcription of eukaryotes & prokaryotes - Interferes with polymerase passage

Question 31

Inhibitors of transcription: death cap

Answer 31

contains α-Amanitin •tightly binds to RNAP II - blocks the elongation step - binds beneath the polymerase’s bridge helix

Question 32

post-transcriptional processing: prokaryotes

Answer 32

not processed much

Question 33

post transcriptional processing in eukaryotes of hnRNA

Answer 33

5' cap polyadenylated 3' end splicing RNA exported from nucleus techinically processed whilst theyre being synthesised

Question 34

RNA capping functions

Answer 34

RNAP contains C terminal domain when phosphorylated it recruits capping enzyme complex the 5' end is modified to 7-methylguanosine (type O cap) joined by 5'-5' triphosphate bridge resist degradation efficient initiation of translation

Question 35

Polyadenylation

Answer 35

influences mRNA stability Complex with Poly(A)-binding Protein (PABP) & prevents degradation.

Question 36

intron consenscous sequences

Answer 36

Found in the “GU-AG” Introns- act as recognition for RNA-binding proteins.

Question 37

how are introns removed

Answer 37

hydrolysis

Question 38

what is needed for splicing

Answer 38

spliceosome –snRNAs and proteins = snRNP

Question 39

why are introns paradoxical

Answer 39

mutations can lead to disease

Question 40

why is splicing needed

Answer 40

can produce different proteins (different destination of cell and activity) by alternative splicing