M1 L6: Transcription

Question 1

What’s the purpose of transcription? Why is it necessary?

Answer 1

Make RNA that’s complementary to DNA. Need to make RNA to make proteins because DNA can’t leave the nucleus if eukaryotic cells

Question 2

What types of RNA don’t code for proteins?

Answer 2

rRNA and tRNA

Question 3

5 differences between RNA and DNA

Answer 3

1) RNA in nucleus and cytoplasm of eukaryotes, DNA only in nucleus

2) RNA has uracil instead of thymine

3) RNA has 2’ OH group, DNA doesn’t

4) RNA is stable as a single strand, DNA is stable as a double helix

5) RNA can form self complementary hairpin loops

Question 4

What are the 3 types of RNA and their functions

Answer 4

rRNA makes up ribosomal subunits

tRNA translates codons of nucleotides into amino acids and proteins

mRNA codes for proteins

Question 5

Which DNA strand is the RNA most similar to?

Answer 5

non template strand (all bases same except U replaces T)

Question 6

how many consensus sequences are in bacteria? where are they? What’s their purpose? Are they variable or conserved? Why?

Answer 6

2

Pribnow box (-10) and -35 consensus sequence (separated by highly variable spacer sequence)

Tell RNA polymerase location and direction of transcription

Highly conserved because mutations would prevent RNA polymerase binding and transcribing

Question 7

What’s the 5’ UTR? 3’ UTR? Termination region?

Answer 7

5’ UTR is region upstream of start codon. Transcribed but not translated.

3’ UTR is region downstream of stop codon. Transcribed but not translated

Region downstream of stop codon that regulates termination of transcription

Question 8

How many RNA polymerases are in bacterial transcription?

Answer 8

1

Question 9

How do bacterial RNA polymerases bind to the promoter?

Answer 9

A sigma factor binds to RNA polymerase –> holoenzyme (complex of subunits with full enzymatic capacity) –> binds to promoter

Question 10

Why do bacteria have multiple sigma factors?

Answer 10

Sigma factors change the specificity of RNA polymerase to bind to different promoters –> bacteria can control which genes get transcribed

Question 11

What are the steps for initiation in bacterial transciption?

Answer 11

1) holoenzyme binds promoter

2) RNA polymerase unwinds ~18bp of DNA starting at (-10)

3) Sigma factor dissociates, RNA starts transcribing at (+1),

Question 12

What are the steps for elongation in transcription?

Answer 12

1) RNA polymerase keeps ~18 bp DNA unwound

2) RNA polymerase adds nucleotides to 3’ end of RNA

3) DNA hydrogen bonds reform behind RNA polymerase

Question 13

Can transcription and translation occur simultaneously in eukaryotes and bacteria? why/why not?

Answer 13

Yes in bacteria because the RNA does not have to leave the nucleus or be processed (cut out introns/5’ cap/poly A tail). No in eukaryotes because RNA has to be processed before it can leave the nucleus and be translated in the cytoplasm

Question 14

What are the two types of termination in bacteria transcription? Which is more common?

Answer 14

Intrinsic and rho dependent. Intrinsic is more common

Question 15

Describe intrinsic termination in bacterial transcription

Answer 15

The termination sequence in DNA is: inverted repeat, spacer, inverted repeat, polyA sequence

When this is transcribed, the inverted repeats complement each other and form a stem loop –> slows RNA polymerase down

The polyA sequence in DNA is loosely bound to the polyU sequence in the new RNA (only 2 hydrogen bonds between the bases) –> RNA polymerase is unstable and dissociates

Question 16

Describe rho dependent termination in bacterial transcription

Answer 16

Rho utilization site in DNA gets transcribed –> rut site in RNA

Rho protein binds rut site and races down the RNA

RNA polymerase slows down because of stem loop, rho crashes into RNA polymerase –> RNA polymerase dissociates

Question 17

How many RNA polymerases are used in eukaryotic transcription? What are their functions?

Answer 17

1) Pol I makes rRNA

2) Pol II makes mRNA

3) Pol III makes tRNA

Question 18

what are 4 differences between eukaryotic and prokaryotic transcription?

Answer 18

1) eukaryotic promoters have more variation (# consensus seqs and locations relative to TSS)

2) molecular apparatus at promoter is more complex in eukaryotes

3) eukaryotic genes have introns and exons

4) eukaryotic DNA is associated with histones which affect amt of transcription

Question 19

what are the 3 ways to investigate eukaryotic promoters?

Answer 19

1) experiment to see which sequences are associate w/ TFs

2) use comparative genomics: look for sequences similar to known promoters in dif genomes

3) do mutational analysis to see how bp mutations affect amt of transcription relative to non mutants

Question 20

What are the common eukaryotic consensus sequences for RNA pol II? What are their locations? Do all promoters have them? What is their purpose?

Answer 20

1) TATA box (-25)
2) CAAT box (-80)
3) GC rich region (-90)

Promoters can have any combination of all or some

Locations can vary (esp. CAAT and GC)

Consensus seq important for binding of transcription factors (proteins that help direct RNA pol and initiate transcription)

Question 21

What are the steps to initiation with RNA pol II in eukaryotic transcription?

Answer 21

1) TBP (TATA binding protein - some genes use TLF) + TAF, = TFIID –> TFIID binds to TATA box –> initial committed complex

2) TFIIA, TFIIB, TFIIF, RNA pol II bind to initial committed complex

3) TFIIE, TFIIH bind to complex –> preinitiation complex (TFIIA, B, D, E, H, F = 6 transcription factors)

4) complex of TFs directs RNA pol II to start transcription at (+1)

Question 22

What are 3 things that can influence transcription regulation in eukaryotes?

Answer 22

1) mutations in promoter usually decrease transcription (random changes in organized entities more likely to decrease performance than increase)

2) how tight DNA is wound around histones (euchromatin: loose –> TFs can bind, more transcription; heterochromatin: tight –> TFs can’t bind, less transcription)

3) enhancer sequences (longer range regulation): activator proteins bind enhancer and co-activator proteins, DNA folds over and activator/co-activator proteins make protein bridge –> interact with other TFs near TSS to enhance transcription

(silencers can work by same mechanism but prevent transcription - way to turn genes off)

Question 23

What are the eukaryotic consensus sequences for RNA pol I? What are their locations? Do all promoters have them?

Answer 23

1) core element (-45 to +20), NEED for transcription

2) upstream control element (-150 to -100), INCREASES transcription

all rRNA promoters have both

Question 24

What are the steps to initiation with RNA pol I in eukaryotic transcription?

Answer 24

UBF1 binds upstream control element, SL1 binds core element –> complex recruits RNA pol I, initiates transcription

Question 25

What are the eukaryotic consensus sequences for RNA pol III? What are their locations? Do all tRNA promoters have them?

Answer 25

Box A (+55) and Box C (+80)

All tRNA promoters have both boxes

Question 26

What are the steps to initiation with RNA pol III in eukaryotic transcription? Why is RNA pol III unique?

Answer 26

1) TFIIIA binds to Box C, TFIIIC binds to Box A

2) TFIIIB binds to both TFIIIA/C

3) RNA pol III binds to complex, starts transcription at (+1)

RNA pol III is unique because it uses an internal control region (downstream of TSS)

Question 27

How is archaean transcription more similar to eukaryotes than bacteria?

Answer 27

Archaean RNA polymerase is more similar to RNA pol II than to bacterial RNA pol

Archaea use TATA box, TFIIB, TBP (archaea only use these proteins)

Question 28

what are the differences between mRNA in bacteria and eukaryotes

Answer 28

1) eukaryotic mRNA has introns and exons

2) eukaryotic mRNA lasts longer and requires processing (5’ cap, poly A tail, splicing)

Question 29

Explain the process of adding the 5’ cap

Answer 29

1) Guanylyl transferase removes the 5’ gamma phosphate from the nucleotide on the 5’ end of the mRNA

2) GT removes the beta and gamma phosphates from a GTP to make a GMP

3) GT joins the phosphates of the two nucleotides, forming a 5’ to 5’ phosphodiester linkage

4) Methyl transferase methylates the 5’ cap and possibly other nucleotides

Question 30

what are the functions of 5’ cap

Answer 30

1) protect mRNA from degradation
2) facilitate transport of mRNA from nucleus
3) facilitate translation by helping ribosome orient itself
4) facilitate intron splicing

Question 31

explain the process of adding the poly A tail

Answer 31

1) CPSF binds polyadenylation signal sequence, CSTF binds U rich region downstream; CFI, CFII, and PAP bind

2) mRNA cleaved 15-30 bases downstream from polyadenylation seq –> releases mRNA bound by CTSF, CFI, CFII

3) PAP adds 20-200 adenine nucleotides, PABII proteins bind to PAP and increase the rate of polyadenylation

Question 32

What are the functions of the poly A tail? Do all eukaryotic transcripts have them?

Answer 32

1) protect mRNA from degradation
2) facilitate transport out of nucleus
3) help orient ribosome for translation

Not all eukaryotes have poly A tails. Some use stem loop structures instead (ex. histones)

Question 33

explain the torpedo model of termination. How is it similar to rho dependent termination? How is it different?

Answer 33

1) 3’ cleavage downstream of polyadenylyl region leaves an uncapped 5’ end of mRNA (RNA pol II still transcribing this strand)

2) torpedo RNAase is specific for this uncapped 5’ end of mRNA and binds to it

3) torpedo RNAase degrades the mRNA until it reaches the RNA pol II, crashes into it, and dissociates it from the DNA –> transcription stops

similar: proteins race down mRNA and dissociate RNA pol from DNA when they collide

dif: rho is in bacteria, torpedo is with RNA pol II in eukaryotes

Question 34

How were introns discovered? Explain how the results led to the conclusions

Answer 34

Hybridizing a single strand of DNA with its complementary mRNA

DNA looped out when there weren’t complementary regions in the mRNA

The regions that complement = exons, regions where there are loops = introns

Question 35

what are the 3 mechanisms of intron splicing?

Answer 35

1) spliceosome
2) self splicing (ribozymes)
3) enzymatic removal of introns in tRNA

Question 36

explain how spliceosomes remove introns

Answer 36

1) snRNP (small nuclear ribonuclear protein) U1 binds to the 5’ splice site, U2 binds to the branch site

2) U4, U5, U6 bind –> forms inactive spliceosome –> mRNA folds into lariat structure

3) U4 dissociates –> spliceosome active –> cleave 5’ splice site

4) lariat structure stabilized by 5’ to 2’ phosphodiester bond between 5’ G and invariant A in the branch site

5) 3’ splice site cleavage

6) exons ligated, lariat structure degraded

Question 37

What 3 regions denote introns

Answer 37

5’ and 3’ splice sites, branch site (20-40 bp upstream of 3’ splice site with invariant A)

Question 38

explain self splicing introns

Answer 38

• are an example of ribozymes (catalytic RNA)

1) exon intron base pairing; a G attacks and cleaves a U-A phosphodiester bond at exon-intron jxn

2) 3’ OH of the U attacks and cleaves the G-U phosphodiester bond at intron-exon jxn

3) intron released, exons ligate

Question 39

Advantage of alternative splicing? How does it work?

Answer 39

can make different proteins from one gene

splicing together different introns/exons

using different promoters (dif TSS)

alternative sites of polyadenylation

Question 40

how is rRNA processing different between bacteria and eukaryotes

Answer 40

bacteria: make 30S rRNA –> needs to get cleaved into subunits, some rRNA genes not in tandem, some tRNAs transcribed too and need to be cut out

eukaryotes: make 45S rRNA, all rRNA genes exist as tandem repeats, tRNAs transcribed individually; ETS, ITS1, ITS2 get cut out (are highly variable and can be used to identify species)

Question 41

how are tRNA genes different in bacteria and eukaryotes

Answer 41

bacteria: some tRNAs transcribed alongside 30S rRNA

eukaryotes: all tRNAs transcribed individually by RNA pol III, NOT alongside 45S rRNA; some tRNAs have introns that get cut out with specialized nucleases