RNA Synthesis and Transcription

Question 1

What chemical reaction is is catalyzed by RNA polymerase?

Answer 1

5’ to 3’ formation of RNA. It catalyzes phosphdiester bonds between ribonucleotides.

Question 2

Why is the RNA polymerase reaction unidirectional?

Answer 2

Because there is a reactive hydroxyl group at the 3’ carbon. The 5’ carbon is stable with a phosphodiester bond.

Question 3

Describe 3 common steps between bacterial and eurkaryotic RNA polymerases in the transcription cycle.

Answer 3

Initiation, Elongation, Termination

Question 4

What are the 3 steps of Initiation?

Answer 4

RNA pol binds to promoter sequence on DNA, pol melts DNA near transcription start site forming transcription bubble, pol catalyzes phosphdiester link of 2 rNTPs

Question 5

What happens during elongation?

Answer 5

Polymerase advances 3’ to 5’ down template strand melting DNA and linking rNTPs

Question 6

What happens during termination?

Answer 6

At transcription stop site, Pol releases completed RNA and disasscoiates from DNA.

Question 7

Name 4 cellular RNA polymerases and their main functions.

Answer 7

E.Coli - transcribes all RNA in E.ColiRNA I [human] - makes rRNARNA II [human] - makes mRNARNA III [human] - makes tRNA

Question 8

What is a promoter?What are common sequence elements in promoters?

Answer 8

A sequence of DNA upstream of the transcription start site where RNA pol binds.TATA - in lots but not all eukaryotic genes, approx 30 base pairs from start

Question 9

How does alpha amanitin block transcription?

Answer 9

Alpha amanitin is a non-competitive inhibitor of RNA pol II. It binds the bridge helix and prevents the polymerase from sliding down the DNA chain.

Question 10

How does rifampicin work?

Answer 10

Rifampicin, a broad spectrum antibiotic, binds the beta subunit of bacterial RNA pol which blocks the RNA exit channel.

Question 11

What are the five components of RNA pol II pre-initiation complex?

Answer 11

TFIIB - places RNA Pol @ correct start siteTFIID - recognizes TATA boxTFIIE - attracts/regulates TFIIHTFIIF - stabilizes RNA pol action, attracts E+HTFIIH - unwinds DNA @ start point, releases RNA pol from promoter

Question 12

What clinical syndromes might one get with a mutation(s) in their TFIIH subunits?

Answer 12

Xeroderma pigmentosum, cockaynes syndrome, TrichothiodystrophyBecause TFIIH functions in transcription and DNA repair

Question 13

What are the three ways that pre mRNA gets processed?

Answer 13

1. Capping - 5’ triphosphate gets replaced by 7-methylguanosine2. Splicing - excision of introns by spliceosome3. Cleavage by endonuclease and polyadenylation at the 3’ end* This all happens while RNA is being made

Question 14

What are the differences between pre-mRNA and mature mRNA?

Answer 14

Pre-mRNA is longer, has a triphosphate group at the 5’ end, and lacks a poly A tail.

Question 15

What are the functions of the 5’ methyl cap?

Answer 15

It makes the 5’ end resistant to exonucleases. It also helps with splicing/processing through a cap binding complex. The cap is recognized for transport to ribosome. Cap removal is signal for degradation.

Question 16

What are the three reactions necessary to add a 5’ cap to pre-mRNA?

Answer 16

1. Remove the 3rd/last phosphate2. Add guanosine triphosphate3. Methylate the 7 position of the guanine cap

Question 17

What is the conserved sequence at the 5’ and 3’ ends at most introns?

Answer 17

5’: GU3’: AG

Question 18

What is the consensus sequence at the poly-A site?

Answer 18

AAUAAA

Question 19

How does alternative splicing permit production of multiple proteins?

Answer 19

Exons can be retained or removed, truncated at the 5’ or 3’ end, mutually exclusive. Plus, introns can be retained.

Question 20

What are two examples of genetic orders caused by splicing defects?

Answer 20

Marfan’s syndrom - mutations that disrupt splicing of fibrillin gene. Which produces protein for walls of heart = weak -> aneurysmsCD44 - cell surface glycoprotein is a predictor of tumor metastasis.

Question 21

What is U1 snRNA?U2 snRNA?

Answer 21

U1 snRNA binds to the GU splice site of intronsU2 binds to the branch point between 5’ and 3’ splice sites.

Question 22

What two reactions make the mature 3’ end of mRNAs?

Answer 22

Cleavage and polyadenylation

Question 23

What is the relationship between 3’ end processing of the pre-mRNA and termination of transcription at the end of a gene?

Answer 23

3’ end formation by cleave/polyadenylation is coupled to termination of transcription by RNA pol II.

Question 24

What are the two major functions of mRNA’s poly-A tail?

Answer 24

Translation + Stability

Question 25

Give an example of how alternative poly-A sites can be used to make more than one protein from a single gene.

Answer 25

A common gene may have multiple AAUAAA sequences.Depending on which poly-A site is used, two different forms of IgM are made: heavy chains and light chains.