Biochemistry Lecture 4 - RNA Synthesis Flashcards Preview

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What are the two primary ways RNA differs from DNA?

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


Why can't RNA form 2x helix?

2' OH - steric hindrance


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



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

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


Which RNA polymerase makes 18S ribosomal subunits?

RNA polymerase I


Which RNA polymerase makes 5.8S ribosomal subunits?

RNA polymerase I


Which RNA polymerase makes 28S ribosomal subunits?

RNA polymerase I


What does RNA polymerase II transcribe?

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


What does RNA polymerase III transcribe?

Small RNAs, tRNA, 5S subunit.


How are the polymerases distinguished in the lab?

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

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


What does RNA polymerase I transcribe?

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


How did eukaryotic RNA polymerases evolve?

Via gene duplication and subsequent sequence divergence.


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

28S, 5.8S, 5S


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



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

tandem arrays


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.

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


How big is the rRNA transcription unit?

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


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

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


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

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


Where are promoters USUALLY located?

Upstream of the gene.


What do enhancers do?

They upregulate transcription.


What are some attributes of enhancers?

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


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

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.


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

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


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



What are the three principal processing steps in mRNA formation?

1. Cap addition
2. Poly adenylation
3. Splicing


What are the purposes of the 5' cap?

Protection against degradation from exonucleases, differentiation.


When is the poly-A tail added?

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


What is a hallmark of mRNAs?

Poly-A tails.


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



Define introns and exons.

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


Describe the splicing steps.

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


Describe the spliceosome.

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.


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

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


What are the Thalassemias?

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.


Define alternative splicing. Name two examples.

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).


What does rifampicin do?

Inhibits bacterial RNA polymerase (inhibits initiation of transcription)


What is Actinomycin D?

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


How is mRNA isolated for cDNA cloning?

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.