Chapter 27 - Transcription

Question 1

Downstream

Answer 1

In the direction of the “current”. 5’ –> 3’. Position # is >0.

Question 2

Upstream

Answer 2

Backwards. 3’ –> 5’. Position# is

Question 3

Template strand

Answer 3

Also the anti-sense strand. Strand that is being used as the template for RNA synthesis.

Question 4

Sense strand

Answer 4

Strand complementary to the template strand. Has the same sequence as the RNA being coded. This sequence makes “sense” to the ribosome.

Question 5

Antisense strand

Answer 5

Strand that is being used as the template for RNA synthesis.

Question 6

Describe prokaryotic promoter structure.

Answer 6

-10 Prifnow box (TATA on sense, untranscribed strand)
17bp spacer (because of σ width)
-35 element

Question 7

How is promoter sequence related to gene expression?

Answer 7

Strong promoter –> recognized well, closer to consensus –> increased transcript amount –> increased gene expression

Question 8

Prokaryotic transcription termination

Answer 8

(1) Sequence dependent (stem-loop)

(2) Protein-factor dependent

Question 9

Sequence dependent termination

Answer 9

(1) GC-rich region causes RNA pol to pause.
(2) Pause allows the GC-region to pair into an RNA stem-loop/hairpin structure
(3) UUUU rich region has weak interactions with DNA (AU bonds), encouraging the DNA and RNA to break apart.

Question 10

Protein factor dependent termination

Answer 10

(1) C-rich sequence signal on the RNA interacts with the ρ factor
(2) Using ATP, ρ moves toward 3’ end of RNA, which is being synthesized
(3) ρ (a RNA-DNA helicase) unwinds the DNA and RNA

Question 11

Polycistronic

Answer 11

Has more than one protein coding region/codes for more than one protein

Question 12

Eukaryotic RNA polymerase I

Answer 12

rRNA transcription

Question 13

What is the most common method of transcription termination in prokaryotes?

Answer 13

Sequence-dependent termination

Question 14

Eukaryotic RNA polymerase I

Answer 14

• rRNA transcription
• UBF-1 and SL-1 (TBP + 3TAFI) transcription factors
• little regulation, always ON
• terminated by steric hindrance
• processed in nucleolus

Question 15

TBP

Answer 15

TATA Binding Protein

Question 16

TAF

Answer 16

TBP-associated factors

Question 17

Eukaryotic RNA polymerase III

Answer 17

• tRNA, 5sr RNA, snRNA
• genes are separate (each with own promoter, many copies)
• 700kDa, 17 subunits

(1) TFIIIA binds first to area downstream of transcription start site
(2) TFIIIIB and TFIIIIC binds to and upstream the TFIIIA, closer to transcription start site
(3) RNA pol III binds to the complex

Question 18

RNA pol IV

Answer 18

Synthesize siRNA and miRNAs

• uncharacterized subunit composition
• transcription THROUGH heterochromatin

Question 19

RNA pol V

Answer 19

Synthesize siRNA and miRNAs

• uncharacterized subunit composition
• transcription THROUGH heterochromatin

Question 20

RNA pol II

Answer 20

Transcribes protein encoding genes (mRNA)

(1) TFIID (TBP and TAFs)
(2) TFIIA stabilizes TFIID-DNA binding
(3) TFIIB recruits…
(4) TFIIF–RNA pol II which recruits…
(5) TFIIE (modulation of TFIIE, increased promoter melting) which recruits…
(6) TFIIH (helicase, promoter melting, promoter clearance)

Question 21

Cis acting element

Answer 21

Element/ regulatory region is on the same molecule as the gene being transcribed/regulated

Question 22

Cis-regulatory elements

Answer 22

Element/ regulatory region is on the same molecule as the gene being transcribed/regulated

Question 23

Element/ regulatory region is on the same molecule as the gene being transcribed/regulated

Answer 23

Cis-acting/regulatory elements

Question 24

Trans-acting elements

Answer 24

Regulatory element is NOT on the same molecule/strand as the gene being transcribed/regulated

Question 25

HDAC

Answer 25

Histone deacetylase, removes acetyl group from histone, making it less charged (and more positive), causing increased DNA-histone interaction

Question 26

HMT

Answer 26

Histone methyl transferase, transfers methyl group to amino group (NH3 ---> N(CH3)3) making it positive charged, bind tightly to DNA

Question 27

Histone acetyl transferase

Answer 27

Adds acetyl group (AcCoA ---> CoASH) to histone, encouraging loose interaction with DNA

Question 28

Chromatin remodelers

Answer 28

Move histones out of the way for access to DNA (uses ATP). Proteins that enable promoter regions to be able to accept the complex/bulky machinery necessary for transcription initiation.

Question 29

Insulators

Answer 29

Prevent TFs from influencing genes next door

Question 30

Polynucleotide phosphorylase

Answer 30

RNAn + NDP ----> RNAn+1 + P2 - not template dependent - adding excess NDP will force the reaction

Question 31

Eukaryotic promoter escape

Answer 31

- After several rounds of 10nt abortive initiation, TFIIH phosphorylates the serine on the C-terminus of RNA polII - TFIIB B-linker opens DNA --> open promoter complex - TFIIB B-reader threads DNA template to active center - When RNA chain promoter escape * TFIIF helps destabilize nonspecific RNA polII-DNA interactions

Question 32

Abortive initiation

Answer 32

RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts that are released before the transcription complex leaves the promotor. 1. RNA polymerase binds to promoter DNA to form an RNA polymerase-promoter closed complex 2. RNA polymerase then unwinds one turn of DNA surrounding the transcription start site to yield an RNA polymerase-promoter open complex 3. RNA polymerase enters into abortive cycles of synthesis and releases short RNA products (up to 10 nucleotides in length) 4. RNA polymerase escapes the promoter and enters into the elongation step of transcription

Question 33

Eukaryotic elongation

Answer 33

(1) Promoter release, core transcription factors are released, elongation begins with helicase clearing the way (2) TFIIF–RNA polII moves along the DNA (3) Elongation factors assist the enzyme in passing through pause sites (e.g. nucleosome remodeling factor, FACT elongation factor)

Question 34

Eukaryotic termination

Answer 34

- RNApolII will usually transcribe well past to the end of the gene (unlike prokaryotic RNA pol) (1) Passes through one or more AATAAA signals past the 3' end of the coding region (2) pre-mRNA (carrying the signal as AAUAAA) is then cleaved by a special endonuclease at 3' end (3) A pol(A) tail is added by a special, nontemplate directed polymerase - mRNA stabilization - facilitation of transport from nucleus to cytoplasm

Question 35

Prokaryotic post-transcriptional processing

Answer 35

(1) Degradation (2) cleavage (3) Non transcriptive nucleotide addition (4) Intron splicing

Question 36

Eukaryotic post-transcriptional processing

Answer 36

(1) Capping (5' methyl capping, GTP added in reverse), position mRNA on ribosome, stabilization of message (2) Splicing (3) Alternative splicing (4) RNA Editing

Question 37

Splicing

Answer 37

(1) U1 snRNP binds to E1 splice site, causing loop with branch site (2) U2 snRNP binds to branch site, causing the strand to fully loop (3) U4/6 and U5 bind (4) With ATP, cleavage occurs at E1 site (U1/U4/U2 vs U6/U5 with the exons) (5) With more ATP, cleavage occurs at E2 site and the exons are joined (6) Ligated mRNA is released

Question 38

Alternative Splicing

Answer 38

Different combinations of exons from the same gene can be processed into different mature mRNAs and then undergo translation into different proteins in different tissues or at different developmental stages of the same organism. hnRNPL is the key player.

Question 39

RNA editing

Answer 39

changes nucleotide sequence of mRNA by changing one base to another

Question 40

Degradation

Answer 40

mRNA degradation (bacteria)

Question 41

Cleavage

Answer 41

pre-rRNA, pre-tRNA --> cleavage to rRNA, tRNA ``` 30S rRNA transcript --> 16S, 23S, 5S tRNA: (1) endonuclease (2) RNAseD removes nt one by one (3) RNase P cuts 5' end (4) RNaseD removes remaining two nt next to CCA sequence (5) Modified bases ```