26: RNA metabolism

Question 1

compare replication and transcription

Answer 1

both require a template, have 5’-3’ direction, initiation, elongation, and termination, and a similar chemical mechanism
transcription does not require a primer, only limited segments of DNA are transcribed and only one DNA strand is transcribed, no proofreading, many RNA copies made from single gene, highly processive

Question 2

three types of RNA

Answer 2

messenger RNA: encode amino acid sequence of polypeptides as specified by a gene
transfer RNA: read the code of mRNA and transfer the appropriate amino acid into the growing polypeptide during protein synthesis
ribosomal RNA: components of ribosomes, the macromolecular protein machines that synthesize proteins

Question 3

compare and contrast DNA/RNA polymerase

Answer 3

RNA polymerase unwinds DNA section forming transcription bubble. transcribes template strand using the same mech as DNA polymerase, 3’ OH acts as nucleophile on incoming ribonucleotide 5’triphosphate. The bubble causes supercoils the topoisomerases deal with. RNA polymerase is less complicated and has no proofreading.
mech slide 4

Question 4

describe the naming conventions of transcription (coding vs template etc)

Answer 4

DNA nontemplate strand is the coding strand because it has same sequence as RNA transcript. coding strand will contain regulatory sequences that affect transcription.
DNA template strand is the strand the RNA polymerase uses to build the RNA transcript. it is complimentary to the transcript
The coding strand of a particular gene can be on either strand and can overlap with genes on the opposite strand (slide 6 if that’s not clear)

Question 5

describe DNA dependent RNA polymerase

Answer 5

uses sigma factor, binds transiently, directs rest of enzyme to specific sites on DNA. different kinds bind different sites

Question 6

describe initiation of transcription

Answer 6

RNA polymerase binds promoters including the -35 region and -10 region (sigma binds) and an upstream consensus sequence elements that is present in genes which are transcribed a lot (alpha interacts?). The upstream elements boost efficiency of RNA polymerase, which depends on sequences, spacing, and distance from start

Question 7

describe DNA footprinting

Answer 7

used to determine what sequence does RNA polymerase bind. Use radioactively labelled DNA fragments (one condition with polymerase present, one without) and treat with DNase so that each strand is cut only once. The strand will not be cut where the polymerase is bound, so by isolating labelled fragments and separating by gel, you will see a gap in the banding where the polymerase was bound and prevented cutting. Tells you the location of the polymerase binding site
slide 9

Question 8

give a general overview of the transcription process in E coli

Answer 8

RNA polymerase core and the sigma factor bind the DNA promoter, forming a closed complex. The transcription bubble forms and an open complex. transcription is initiated, promoter clearance is followed by elongation. elongation continues, sigma factor dissociates and is replaced by NusA. Transcription is terminated as NusA dissociates and the RNA polymerase is recycled

Question 9

two types of transcription termination in E coli

Answer 9

rho independent: RNA polymerase encounters a terminator sequence and an RNA hairpin forms at a palindromic sequence, reducing the length of the RNA-DNA hybrid. The hairpin causes mRNA to release
rho dependent: the rho helicase binds to a rut site and the helicase migrates alone the mRNA to the elongating RNA polymerase. the rho separates the mRNA from the DNA template when it reaches the polymerase, releasing mRNA

Question 10

describe the three eukaryotic RNA polymerases

Answer 10

I: makes pre-rRNA, precursors for 18S, 5.8S, and 28S ribosomal RNA
II: makes mRNAs, some specialized RNAs, and can recognize a variety of promoters (TATA box common)
III: make tRNAs, the 5S rRNA, and some specialized RNAs

Question 11

things to know about eukaryotic RNA Pol II

Answer 11

it needs many other proteins to work and has 12 subunits, its more complex than the prokaryotic version. Each step (assembly, initiation, elongation, and termination) is associated with specific proteins
Has special C terminal domain (CTD) containing many repeats. this can be posttranvslationally modified for signaling and such

Question 12

steps in eukaryotic transcription

Answer 12

Lots of proteins assemble on the DNA, especially note TATA binding protein (TBP) and TFIIH (which has helicase and kinase activity) and Pol II. The closed complex is opened by DNA unwinding via TFIIH helicase activity. Then the open complex undergoes phosphorylation of Pol II (by CDKs) on the CTD, initiation, and promoter escape. The phosphorylation patter on the CTD changes through the process of initation, elongation, and termination. Elongation proceeds with elongation factors associating and termination ends to process, freeing Pol II to be recycled
slide 17

Question 13

what is TFIIH

Answer 13

transcription factor IIH
has helicase and kinase activities important for transcription. also used in nucleotide excision repair (more efficient repair within genes that are actively being transcribed). So as Pol II comes upon damaged Dan and halts, TFIIH can recruit the rest of the DNA repair enzymes for form the complex required to fix the damage

Question 14

how is the amount of transcription controlled

Answer 14

activators: help initiate complex formation. Includes things like transcription factors
repressors: bind to sites and block transcription faction and polymerases

Question 15

how is RNA processed

Answer 15

both eukaryotes and prokaryotes process transcripts by altering bases and sugars (tRNA)
eukaryotic: 5’ capping, splicing, 3’ polyA tail

Question 16

5’ cap details

Answer 16

protects from nucleases, binds to specific proteins and helps the binding of the message to the ribosome. Originates from GTP, methyl groups (SAM), and occurs early. As mRNA is synthesized, the 5’ end associates with the CTD of Pol II and its cap-synthesizing complex which is then replaced by cap binding complex (CBC) which binds and keeps the cap attached to the Pol II
Can you draw a 5’ cap? slide 20

Question 17

how are introns spliced? 4 classes

Answer 17

Group I are self splicing, no proteins involved and no ATP-ribozymes. bacteria and viruses (possibly eukaryotes)
Group II use specialized proteins (snRNPs) and ribozymes
Spliceosomal introns are nuclear primary transcripts spliced via the spliceosome, uses ATP (not for cleavage), some RNAs, and often a lariat mech (snRNPs)
tRNA splicing: requires ATP and an endonuclease, some RNAs

Question 18

group I introns splicing mech

Answer 18

slide 22-23
3’ OH of guanosine (an exogenous nucleophile) acts as nucleophile on the phosphate linking the exon and intron (5’ splice site). forms a bond with the 5’ of the intron, allowing the 3’ OH of the exon to act as a nucleophile at the other end of the intron. releases the intron and leaves a spliced RNA containing only exon material

Question 19

group II intron splicing mech

Answer 19

slide 24
the 2’ OH of a specific adenosine in the intron (internal nucleophile) acts as nucleophile in attacking the 5’ splice site, forming lariat. lariat intermediate has an adenosine with 3 phosphodiester bonds. the now exposed 3’ OH of the 5’ exon acts as a nucleophile on the other end of the intron, completing the reaction

Question 20

describe spliceosomes and their mech

Answer 20

spliceosomes have the same lariat forming mech as group II, but are larger complexes of proteins with RNAs. snRNPs (small nuclear ribonucleoproteins) and snRNAs (small nuclear ribonucleic acids) position substrate to active site of the spliceosome. the intron is bracketed by GU and AG sequences.
spicing can be coordinated with transcription by the spliceosome docking on the CTD and splicing introns as the mRNA is transcribed
slide 26

Question 21

function and process of the poly A tail

Answer 21

binding site for specialized proteins, protects mRNA from degradation (in prokaryotes a poly A tail increases degradation)
process: associates with CTD. cleavage sequences recognized by endonuclease, cleaves off some of the mRNA after the AAUAAA or GU rich sequence (signals the end). polyadenylate polymerase adds A residues (no template required, does need RNA primer)

Question 22

does one gene always code for the same single protein?

Answer 22

no. one gene can be translated and then differentially spliced to create many RNAs and many protein products. there can also be multiple poly A tail sites which give rise to multiple products

Question 23

differential RNA processing example of calcitonin gene

Answer 23

in the thyroid, the calcitonin gene in rats is spliced and ultimately encodes for the product calcitonin. in the brain, the gene is spliced and ultimately encodes for CGRP

Question 24

do rRNAs and tRNAs need processing?

Answer 24

yes. they are often methylated, cleaved, and degraded at specific sequences. tRNA is processed by base modifications, 5’ cleavage, 3’ cleavage, and the addition of a 5-CCA-3 sequence on the 3’ end. some eukaryotic tRNAs have introns

Question 25

review the bases, draw them!

Answer 25

idk google

Question 26

what determines the concentration of a gene product

Answer 26

the rate of mRNA synthesis and degradation. The average half life of vertebrate mRAN is about 3 hours with capping and poly A tail. miRNAs target and degrade mRNAs.

Question 27

describe how miRNAs work

Answer 27

pri-miRNA is formed via transcription (Pol II) of its gene, it has long repeating loops and normal mRNA processing things (cap and tail). Drosha enzyme trims pri-miRNA into Pre-miRNA. Exportin-5 and Ran allow export into cytoplasm. In the cytoplasm, Dicer cleaves the Pre-miRNA and RNA helicase unwinds. The mature miRNA is loaded onto RISC. the RISC miRNA complex selects a target with near perfect complementarity to the miRNA and signals cleavage of the target, halting protein production. If partial complementary is found, the mRNA target is bound but not cleaved, protein production is repressed
slide 37

Question 28

describe RNA dependent synthesis of RNA (Sars2-Cov life cycle)

Answer 28

viral particle is endocytized after spike proteins interact with our membrane. Once inside, releases RNA material (plus sense: can be used directly by our ribosomes to produce protein) which is translated. Viral polymerase is translated, which is an RNA depended RNA polymerase (RNA replicase). Makes a whole series of replications of the RNA (negative sense) which are used to make more plus sense and can be repackaged and leave the cell to spread.
Key thing is the RNA dependent RNA polymerase (RNA replicase)! The viral RNA that is released is initially harmless, but once it gets translated we get problems. RNA dependent = RNA is the template. RNA polymerase = it synthesizes RNA

Question 29

describe RNA dependent synthesis of DNA (retrovirus)

Answer 29

Retroviruses (like HIV) invade cells and release RNA along with reverses transcriptase. RT synthesizes DNA from the viral RNA. The viral DNA can then be integrated into host genome and transcribed/translated into proteins and a new retrovirus
Reverse Transcriptase is an RNA dependent DNA polymerase. RNA dependent = RNA is the template. DNA polymerase = it synthesizes DNA

Question 30

typical components of retrovirus

Answer 30

genes gag, pol, and env are in all retroviruses. Long terminal repeats also consistently present. Pol gene encodes the protease needed to cleave the gene product into Integrase, Protease, and Reverse Transcriptase. Just 3 genes are used to make 8 needed proteins for multiplication

Question 31

what is the gene that relates viruses and cancer?

Answer 31

Src gene (in Rous sarcoma virus genome) encodes Src kinase. The kinase interferes with phosphosignaling and causing constant division (cancer). Src is an oncogene

Question 32

why is it hard to develop anti-HIV therapeutics?

Answer 32

HIV uses reverse transcriptase to infect and multiply. RT is very error prone and so makes different molecules and can adapt/change rapidly to treatments. Must use a whole cocktail of drugs to hit many different fronts simultaneously

Question 33

what are the current inhibitors of HIV RT

Answer 33

AZT and DDI. taken up by T cells and competes with dTTP in reverse transcriptase active site. It does not have a 3’ OH and so when it’s incorporated, DNA synthesis stops.

Question 34

what is telomerase? how does it work

Answer 34

telomerase is a specialized reverse transcriptase (RNA dependent DNA polymerase). it is used to solve the end replication problem where the lagging strand is not replicated all the way to the end due to the need for primers. telomeres (which do not encode genes) are present at the end of chromosomes, they shorten with time. telomerase functions to lengthen the telomeres after replication
telomerase has an RNA template that is kept bound. it then matches up with the DNA strand and builds on the strand using its RNA as a template. DNA polymerase fills in the other strand after RNA primer is placed and ligase seals the nick. the end is still staggered and the exposed single strand is protected by telomere binding proteins