Biochemistry Lecture 4 - RNA Synthesis

Question 1

What are the two primary ways RNA differs from DNA?

Answer 1

Ribose is the sugar (no 2’ OH), thymine is replaced by uracil.

Question 2

Why can’t RNA form 2x helix?

Answer 2

2’ OH - steric hindrance

Question 3

Can RNA loop around and complimentary base-pair with itself?

Answer 3

Yes.

Question 4

The “coding” DNA strand is the one that is used for peptide formation. Which strand is transcribed to RNA?

Answer 4

The non-coding (-) strand is transcribed to make a RNA strand identical to the coding DNA strand.

Question 5

Which RNA polymerase makes 18S ribosomal subunits?

Answer 5

RNA polymerase I

Question 6

Which RNA polymerase makes 5.8S ribosomal subunits?

Answer 6

RNA polymerase I

Question 7

Which RNA polymerase makes 28S ribosomal subunits?

Answer 7

RNA polymerase I

Question 8

What does RNA polymerase II transcribe?

Answer 8

Pol II carries out transcription for genes that code for cellular proteins (heterogenous nuclear RNA, or pre mRNA)

Question 9

What does RNA polymerase III transcribe?

Answer 9

Small RNAs, tRNA, 5S subunit.

Question 10

How are the polymerases distinguished in the lab?

Answer 10

By their sensitivities to alpha-amanitin (derived from poisonous mushroom).

Pol I - resistant
Pol II - sensitive
Pol III - intermediate

Question 11

What does RNA polymerase I transcribe?

Answer 11

rRNA (nucleolar) - EXCEPT the rRNA 5s subunit (pol III does that)

Question 12

How did eukaryotic RNA polymerases evolve?

Answer 12

Via gene duplication and subsequent sequence divergence.

Question 13

Which rRNAs associate with the eukaryotic large (60S) subunit?

Answer 13

28S, 5.8S, 5S

Question 14

Which rRNAs associate with the eukaryotic small (40S) subunit?

Answer 14

18S

Question 15

How are the genes that code for the pre-rRNA arranged?

Answer 15

tandem arrays

Question 16

For RNA pol I, name the factors that recognize and bind the regulatory region (aka promoter) of DNA before RNA polymerase I can come in to start transcription.

Answer 16

Factor B binds first, then S factor comes in, then RNA polymerase I can join.

Question 17

How big is the rRNA transcription unit?

Answer 17

13,700 nucleotides long - transcribed as a single unit.

Question 18

In the gene that codes for the 5S rRNA subunit, where does the promoter region lie?

Answer 18

In the body of the gene (downstream from where transcription will start and migrate away from).

Question 19

What are the transcription factors called that bind to the promoter region of the gene that transcribes for the 5s rRNA subunit?

Answer 19

TFIIIA, B, and C. ( remember III for Polymerase III)

Question 20

Where are promoters USUALLY located?

Answer 20

Upstream of the gene.

Question 21

What do enhancers do?

Answer 21

They upregulate transcription.

Question 22

What are some attributes of enhancers?

Answer 22

They can be at far distances away from the gene, they can be upstream OR downstream, they function in any orientation.

Question 23

The promoter contains the TATA box. What binds to the TATA box? Where is is located?

Answer 23

Basal transcription factors bind. In the case of RNA pol II-catalyzed transcription of pre-mRNA, TATA binding protein (TBP, a subunit of TFIID) binds. It is located approximately 25 nucleotides upstream from the gene.

Question 24

What happens after TATA binding protein in TFIID binds to the TATA region of the promoter?

Answer 24

More TFs are recruited, then RNA Polymerase II comes in, gets phosphorylated, and transcription can begin.

Question 25

Is the binding of basal transcription factors required for transcription of all pre-mRNA genes?

Answer 25

Yes

Question 26

What are the three principal processing steps in mRNA formation?

Answer 26

1. Cap addition 2. Poly adenylation 3. Splicing

Question 27

What are the purposes of the 5' cap?

Answer 27

Protection against degradation from exonucleases, differentiation.

Question 28

When is the poly-A tail added?

Answer 28

During transcription, in the middle of the nascent strand of mRNA!

Question 29

What is a hallmark of mRNAs?

Answer 29

Poly-A tails.

Question 30

What is the nucleotide sequence located 10-30 nucleotides upstream of mRNA poly-A tails?

Answer 30

AAUAAA

Question 31

Define introns and exons.

Answer 31

Introns get snipped out | Exons stay. They are thought to code for individual functional domains of proteins.

Question 32

Describe the splicing steps.

Answer 32

1. Cleavage of 5' site and formation of lariat (loop) | 2. Cleavage of the 3' site and concomitant joining of the two exons.

Question 33

Describe the spliceosome.

Answer 33

Various snRNPs make up the spliceosome. They interact with Adenosine in the intron to form the lariat, and catalyze 3'OH nucleophilic attack on the exons for exon joining.

Question 34

What are the characteristic nucleotide residues on the 5' and 3' ends of introns, respectively?

Answer 34

GU on 5' intron, AG on 3'. Spliceosomes contain some RNA and are thought to recognize these.

Question 35

What are the Thalassemias?

Answer 35

They are hereditary hemoglobin abnormalities that result in either beta of alpha hemoglobin deficiency. 25% of the mutated genes in the Thalassemias are related to RNA splicing.

Question 36

Define alternative splicing. Name two examples.

Answer 36

A single pre-mRNA can be spliced in different ways yeilding different mature mRNA and thus different proteins. Alpha-tropomyosin is one example. Another is the IgGs, some antibodies have PM regions, and some don't (the PM region would be coded for by an additional exon).

Question 37

What does rifampicin do?

Answer 37

Inhibits bacterial RNA polymerase (inhibits initiation of transcription)

Question 38

What is Actinomycin D?

Answer 38

It intercalates in DNA (especially binds to G residues), inhibiting translation. Mammalian cells sensitive to this.

Question 39

How is mRNA isolated for cDNA cloning?

Answer 39

Because we know that poly-A tails are a hallmark of mRNA, we can use oligo dT columns to grab the tails along with the mRNA. Then, deoxythimidine oligos can anneal to the A tails, which act as primers for reverse transcriptase, which will make a cDNA clone. The clones can be put into bacterial vectors for libraries.