MCBI Protein RNA Metabolism
Dr. Van Oost's Summary Slides, in Question Form...
1.
The DNA _________ strand is identical to the mRNA transcribed from the ____________ strand. Why?
The DNA CODING strand is identical to the mRNA, except T is replaced with U. This is because mRNA is made complementary (and antiparallel) to the TEMPLATE strand, which in and of itself is complementary and antiparallel to the CODING strand.
2.
How is 5'-3' directionality reflected in protein directionality?
The ribosome always translates mRNA in the 5'-3' direction, so the protein amino terminus is like the 5', and the carboxyl terminus is like the 3' end. Later, different things will be added to these ends.
3.
Bacteria has __________ RNA polymerase. How many do eukaryotes have? Which are they?
One RNA Pol for bacteria; three for eukaryotes. These are I, II, and III. More info below.
4.
Where does RNA Polymerase begin? What does it need to start there?
RNA polymerase does not need a primer, but can only start at promoter sequences.
5.
What is the role of the promoter in transcription?
These promotor sides tell the RNA polymerase where to bind. Think about the region from -70 to +30. ***RNA polymerase catalyzes attack of the 3' OH of the nucleotide of the growing 5' alphaphosphate.
6.
What does “+1” mean in transcription? Upstream? Downstream? What happens in either direction?
+1 is the start site. No zero. Upstream is before +1 (like the -10, -35 sites), Downstream is after + in the direction the Pol is going.
7.
What does the promoter consist of?
The promotor is a “mosaic of consensus sequences and non-consensus sequences.”
Specifically, the -35 region and the -10 region (aka Pribnow box). This area has a lot of A-Ts.
***Sigma 70 binds the consensus sequence here.
8.
What factors determine the strength of a promoter?
The actual consensus sequences, the spacing between consensus sequences, and the distance of the consensus sequences from the transcription site. Think of these sequences like buttons....do the buttons match the button holes? How far apart are the buttons? How far away are the buttons from the the parts of whatever you're buttoning that you really want to stay matched up?
9.
Over how many bp does the transcription bubble extend? The RNA/DNA hybrid?
15-17 bp, 7 bp
10.
How is the termination of transcription regulated (what sequences...)?
For Rho-dependent, Rho binds to CA-rich regions upstream from a termination site, then races to catch up to the RNA polymerase to stop it and dissolve everything. With Rho-independent, it is the weak UA binding in the RAN hybrid that is pulled apart when there is a lot of GC binding in the newly formed
RNA....when it forms a hairpin, it's too heavy for the weaker UA bonds to stay attached, so they break and transcription stops.
11.
How does rifampin work? What is it often used to treat? What does actinomycin do?
Used for TB (and others), rifampin binds to prokaryote RNA Pol to change its conformation to prevent transcription. Actinomycin binds to dsDNA so it can't open up well for

transcription. At high dose, it stops it altogether.
12.
What are the three eukaryotic polymerases, and what classes of RNA do they synthesize?
Yeah, just know this chart. Step I likes to ask about amanitin, and you need to know what the cellular transcripts and locations are, anyway.
I.
Nucleolus 18S, 5.8S, and 28S Insensitive to amanitin
II.
Nucleoplasm mRNA precursors, snRNA and miRNA amanitin
STRONGLY inhib. By
III.
Nucleoplams tRNA and 5S rRNA Inhibited in high concentrations by Amanitin
Also, remember that Euk. RNA Pol II has 12 subunits, and one of those has a long CTD
(carboxy terminal domain), with 52 YSPTSPS repeats. Serine is the important one there.
13.
What precedes the binding of eukaryotic RNA Polymerase II to the promotor? How do those elements work together?
The binding of euk. RNA pol II to the promotoer is preceded by the binding of transcription factors, since it can't start on its own, like prokaryote RNA pol. TFIIX is a general name, for Transcription
Factor II (for RNA pol II) X (for add something here later).
In terms of the steps: Starts with TBP binding to TATA box, then TFIIB binds to BRE and
TFIID/TBP. THEN, RNA Pol II can bind, followed by other TFIIs. After it's done, RNA Pol II is recycled by dephosphorylation.
14.
What are the functions of TFIIH?
Has a helicase activity to open up the DNA and a kinase activity.....is involved in making the C-terminal regon of the RNA polymerase a binding site for enzymes carrying out capping, splicing, and polyadenylation. Spcifically, TFIIH targets the Serine residues on that YSPTSPS tail in the CTD.
15.
What all is involved in the 5' cap?
RNA processing requires a variety of small RNAs and their associated proteins (snRNAs), which, with other enzymes carries out capping and polyadenlyation. This is in eukaryotes....bacteria use cotranslation, so they're transcripted at the same time....no chance for processing.
So, for the 5' cap, a methylated Guanosine in a 5'-5' phosphotriester binds to the 5' end of the new/nascient mRNA. This is carried out with the enzyme guanylyltransferase. Methylation at N7 is carried out in the cytosol to create this methylated guanosine. This protects the mRNA from degradation and is important for translation initiation.
16.
What is polyadenylation? What is it also called? What initiates it?
Poly A tail. It's the replacement of the 3' end of the new mRNA by a polyA tail, started by the appearance of the polyadenylation signal AAUAAA. Then, up to 200 As are added. These are truly add-ons, as they are not coded for in the DNA. The AAUAAA site is cleaved by the specific endonuclease, and then polyadenylate polymerase actually adds the polyA tail.
17.
What is splicing? How does it work? What are the snRNPs and what do they do?
The removal of introns in the nucleus, so only the exonic mRNA that will actually code for something is sent out in the final copy. The snRNPs are the U1-U6 proteins that help the splicing process along. U1 binds to before the splice donor site, and U2 binds to the adenosine on the branch site, and that 2'-OH binding activates the site to attack the phosphodiester bond from the donor site, making a lariat. There, U4 brings in U5 and U6. U5 forms a bridge at the base of the lariat bring the two ends of the intron together. U6 separates and joins to U2, and then U2 and U6 catalyze the

transesterifications to break the bonds to let the intronic lariat break off.
18.
What is mRNA degradation important for?
Regulation of mRNA concentration within the cell. In bacteria, mRNA halflife is a few minutes; in eukaryotes, can be a few minutes or up to a day. Without degradation, the mRNA concentraion would be out of control.**Bacterial mRNA is degraded by an endoribonuclease followed by 3' to 5' exoribonuclease action.
19.
What happens when the Poly A tail is shortened to less than 25 A's? In which direction?
The RNA starts degrading from both the 3' and 5' ends. This is followed by decapping.
20.
How are ribosomal RNA genes first transcribed? Then what happens to them?
They are first transcribed as a cluster, then methylated and cleaved by Rnases into the mature products. These rRNAs are stable, have a “Svedberg number” based on size, are very complex with
Watson-Crick duplexes.
21.
What are modified bases, and what RNA has a lot of them? Why does this matter?
They are modified bases in tRNA, which allow for better recognition in loops. Inosine in the anticodon allows for the recognition of multiple codons.
22.
What happens to the primary transcript of tRNA at the site where amino acids will attach?
In eukaryotic tRNA, the 5' end is removed with RnaseP, a small intron is spliced out, and a CCA sequence is added to the 3' end that was cut by RnaseD.
23.
What are microRNAs? What is RISC?
These are transcribed by RNA pol II and are capped/polyadenylated in the nucleus and cytoplasm to short, single-stranded RNAs that are loaded onto RISC, the RNA induced Silencing
Complex. These are cut with Dicer.
24.
How do reverse transcriptases work? What's an example of a virus with this activity?
They make double stranded DNA from an RNA. Retroviruses do this. No proofreading! They work by 1) RNA-dependent DNA polymerase activity, 2) RNA degradation from the RNA/DNA hybrid, like RNase H, and 3) DNA-dependent DNA synthesis with DNA pol activity.
25.
How does telomerase work, and why should we care?
Telomerase is a reverse transcriptase that adds telomeric repeats at the end of a chromosome to protect it from degradation. Each time DNA gets replicated, the end shortens on the lagging strand because the polymerases needed a place to latch onto and start. Telomerase adds onto the 3' end so that the polymerase has a place to hold onto and start working, without taking up the coding information site.
Like adding a zipper extension to one side of a hoodie zipper to make sure that it can zip all the way up to the top.