Exam 2 - Gene Structure and mRNA Transcription

Question 1

Do we know what each base in the genome is doing?

Answer 1

Protein coding = 40% w/ introns.

2% w/o introns

Question 2

gene

Answer 2

Entire nucleotide sequence that is necessary for synthesis of a functional gene product (RNA/protein)

Introns, exons, regulatory sequences

Question 3

Enhancers

Answer 3

may be upstream or downstream (or in introns) and as far away as hundreds of kilobases from the transcription start site.

Question 4

Promoter-proximal elements

Answer 4

can be both upstream and downstream

influence the rate of transcription. Position RNA polymerase II to initiate transcription at the start site.

Question 5

Cis and trans-regulatory elements

Answer 5

Cis = sequences that impact transcription

trans = proteins that bind and impact transcription

Question 6

TATA box

Answer 6

Core promoter element TATATATA

TATA-binding protein binds (TBP)

Highly conserved

NOT TRANSCRIBED

Question 7

How can we find TATA boxes in GOI?

Answer 7

Serial deletion/mutagenesis can identify important/necessary promoter regulatory sequences.

Question 8

RNA polymerase II know where to land

Answer 8

RNA most be decondensed

TATA Box recruits RNA pol II with general transcription factors

Question 9

3 stages of transcription

Answer 9

Initiation
Elongation
Termination

Question 10

Initiation

Answer 10

RNA polymerase II melts DNA duplex to form a transcription bubble.

Begins polymerizing (rNTPs) at start site

Question 11

Elongation

Answer 11

Polymerase advances

5’ end of RNA strand displaced from template DNA and exits through channel in RNA polymerase II

Question 12

Termination

Answer 12

RNA polymerase II dissociated from template DNA at a specific termination site.

Question 13

Preinitiation complex

Answer 13

General transcription factors

Gene-specific transcription factors

Mediator: Allows activator proteins to communicate properly, correctly position TFIIH

Distal enhancers act via looping DNA

Question 14

Overall Mechanism

Answer 14

1.) TATA-binding protein binds and bends DNA (within TFIID which is like a saddle)

2.) Other general transcription factors and RNA pol II follow + bind sequentially to the same region

3.) TFIIH helicase uses ATP hydrolysis to unwind the DNA at the transcription start site. Forms transcription bubble

4.) Pol II helps melt DNA at the transcription start site, forming bubble

5.) Pol II initiates transcription in resulting open complex

6.) TFIIH kinase domain phosphorylates pol II CTD

7.) General transcription factors dissociate from promoter

8.) Poll II continues transcription away from promoter

9.) RNA polymerase stops at the termination site

Question 15

1.) TATA-binding protein (TBP) binds

Answer 15

Bends DNA providing a physical landmark

Brings proteins on both sides closer

Question 16

2.) Other general transcription factors and RNA polymerase II follow and bind sequentially to the same region

Question 17

3.) TFIIH helicase uses ATP hydrolysis to unwind the DNA at the transcription start site. Forms transcription bubble

Answer 17

uses ATP

Question 18

4.) Pol II helps melt DNA at the transcription start site, forming bubble

Question 19

5.) Pol II initiates transcription in resulting open complex
6.) TFIIH kinase domain phosphorylates pol II CTD

Answer 19

Transcribed 5’ to 3’

RNA polymerase II catalyzes formation of phosphodiester bond to form nucleotide chain

Question 20

7.) General transcription factors dissociate from promoter

Answer 20

Kinases module phosphorylates RNA polymerase II on serine 5, becomes docking site for mRNA capping enzymes

General TF dissociate, elongation starts.

Question 21

8.) Poll II continues transcription away from promoter

Answer 21

Pauses 50-100 bp to allows emergency proteins such as HSP70

Allows time for capping enzymes to associate CTD tail of RNA polymerase II

Allows multiple RNA polymerase II to line up and transcribe at once with HSP

Question 22

Nelf

Answer 22

Negative elongation factor

Plugs rNTP flowing channel to pause elongation

Question 23

9.) Termination

Answer 23

RNA pol I requires specific 18-nucleotide sequence recognized by termination protein

RNA pol II in eukaryotes takes place 1,000-2,000 nucleotides beyond end of gene being transcribed

RNA pol II needs mRNA hairpin (similar to termination of transcription in prokaryotes)

Question 24

RNA processing & post-transcriptional gene control

Answer 24

1.) GTP 5’ Cap
2.) Cleavage at Poly A site
3.) PolyA polymerase add poly-A tail (polyadenylation)
4.) RNA splicing, spliceosomes
5.) Translation

Question 25

hnRNPs (RNA binding proteins)

Answer 25

keep mRNA linear to fit through NPC without tangling

Question 26

mature, functional mRNA components

Answer 26

5’ cap
5’ UTR
Exon
3’ UTR
Poly-A tail

Question 27

Inverted structure of 5’ capping

Answer 27

5’ to 5’ linkage
stabilizes mRNA
Aids in nuclear transport
Key role in translational initiation

Question 28

Intron splicing

Answer 28

spliceosome into lariat shapes

Premature = stay in nucleus
Nuclear exosomes complex degrades

Question 29

Alternative splicing

Answer 29

Creating diversity

Production of different proteins from same gene

Question 30

mRNA in cytosol

Answer 30

Nonsense mediated decay

Question 31

microRNA

Answer 31

small non coding double stranded RNA

RNA silencing and gene expression as post-transcriptional regulators

Can check for knockdown efficiency with western blots