RNA Metabolism Flashcards
Is the coding strand identical or complementary to the mRNA (except for T/u)?
The coding strand is identical
Is the DNA template strand complementary or identical to the coding strand?
DNA template strand is complementary and antiparallel to the mRNA
In what direction is mRNA synthesized?
5’ to 3’ from the template strand
In what direction do ribosomes translate mRNA?
In the 5’ to 3’ direction, synthesis goals the protein from the amino terminus to the carbonyl terminus
When the template strand is copied into RNA, what strand is it on the DNA helix?
The 3’ to 5’ strand in the DNA double helix (the “bottom strand” in written DNA)
What strand is the coding strand?
The non-template strand (the “top strand” in written DNA)
Can be read as the RNA strand provided all T’s are read as U’s
How many RNA polymerase are found in bacteria?
Only 1 type
Does RNA polymerase have proofreading ability?
Nope, it makes errors at a rate of 1 in 10k bases
When can RNA polymerase start transcription?
At a particular DNA sequence in front of the gene (promoter)
It does not require a primer
Where does RNA polymerase bind when the promoter or promoter site is found?
From 70 bp’s before the transcription start site to 30 bp after the transcription start site
What is the sigma factor?
The part of the promoter site which binds to RNA polymerase.
- a detachable subunit of RNA polymerase and guides it to the promoter
What is the combination of the core enzyme (RNA polymerase) and the sigma factor called?
The holoenzyme
What drives the reaction of RNA polymerase?
Hydrolysis of pyro-phosphate drives the reaction
Where does RNA polymerase attack in order to grow an RNA chain?
RNA polymerase catalyzes an attack on the 3’ OH of the nucleotide at the end of the growing strand, using the 5’ alpha-phosphate of the incoming NTP
How do you denote the DNA sequence of a transcribed DNA?
The first nucleotide (start site) of a transcribed DNA sequence (coding strand) is denoted as +1 and the second one as +2 etc
The nucleotide preceding the start site is -1
What is the pribnow box?
The -10 region in a bacterial promoter sequence
What are the two conserved promoter sequences found in bacteria?
- 35 region
- 10 region, also called the pribnow box
How does RNA polymerase determine orientation of promoter sequence?
An RNA that moves from left to right makes RNA by using the bottom strand as a template
An RNA polymerase that moves from right to left makes RNA by using the top strand as template
what are the 3 factors important for promoter strength?
- The consensus sequences
- The spacing between the consensus sequence
- The distance of the consensus sequences from the transcription start site
What can increase efficiency of the promoters?
Negative supercooling by topoisomeases can increase efficiency of promoters
After the promotor is cleared and RNA polymerase attaches what happens?
A major conformational change that cannot revert
How many Bp remain unwound during transcription?
15-17 bp remain unwound during transcription (= transcription bubble) and RNA/DNA hybrid stays limited to 7bp
What are the two termination of bacterial transcription?
- Rho factor
2. Rho factor-independent
What is the Rho factor?
An ATP-dependent helicase which can unwind RNA/DNA hybrids
Where does the Rho factor attach to and what does this do.
The Rho factor bind to a CA-rich region in the nascent RNA chain some distance upstream of the termination site and then catches up with the RNA polymerase (transcription bubble) and dissolves the mRNA-DNA-polymerase complex (RNA strand).
What is the Rho factor independent termination of bacterial transcription?
Terminated by a combined action of weak U-A bonding in the RNA/DNA hybrid and strong G-C bonding in the nascent RNA
I.e the hairpin of the nascent RNA; the RNA fold on itself
What does Rifampin do?
Binds to RNA polymerase and changes its conformation so that it cannot initiate RNA synthesis.
RNA polymerase from eukaryotes cells does not bind rifampin, and RNA synthesis is unaffected
What is actinomycin?
An antibiotic that binds strongly to double stranded DNA (prevents it from being an effective template for RNA synthesis)
What happens when you have high concentrations of actinomycin?
Inhibits DNA replication
It’s specificity for double stranded DNA can be explained by intercalation
What doe the antibiotic rifampin inhibit?
Initiation of transcription by binding to the RNA polymerase
What is the location of RNA polymerase in I, II, III?
I: Nucleolus
II: Nucleoplasm
III: Nuceloplasm
What are the cellular transcripts of eukaryotic RNA polymerase I, II, III?
I: 18s, 5.8s and 28s rRNA
II: mRNA precursors, snRNA and miRNA
III: tRNA and 5S rRNA
What are the effects of alpha-amanitin (from mushrooms) on the Eukaryotic RNA polymerase?
I: insensitive
II: strongly inhibited
III: inhibited by high concentrations
What part of the Eu RNA polymerase differ?
The C-terminal part, which is substantially longer for RNA pol II
What is alpha-amanitin?
A poison found in mushrooms
How many subunits does the bacterial RNA polymerase have?
5
What does Eukaryotic RNA polymerase II require for it to begin transcription?
“General transcription factors” (TFIIX)
What is DPE?
What’s the consensus sequence and transcription factor?
Downstream promoter element
Consensus: A/G G A/T C G T G
General transcription factor: TFIID
What does TFIID do?
TBP subunit (1): recognizes TATA box
TAF subunits (~11): recognizes other DNA sequences near the transcription start point; regulates DNA-binding by TBP
What does TFIIB do?
Recognizes BRE element in promoters
Accurately positions RNA polymerase at the start site of transcription
What does TFIIF do?
3 subunits
Stabilizes RNA polymerase interaction with TBP and TFIIB; helps attract TFIIE and TFIIH
What does TFIIE do?
How many subunits
2
attracts and regulates TFIIH
What does TFIIH do? How many subunits?
9 subunits
Unwinds DNA at the transcription start point
Phosphorylates Ser5 of the RNA polymerase CTD
Releases RNA polymerase from the promoter
What is CTD?
Carboxy-terminal domain
When are most of the transcription factors released by after the complex starts.
60-70 nucleotides (nt)
How is RNA polymerase recycled in eukaryotes?
Dephosphorylation
What does RNA polymerase require for removal of the positive supercoils in front of it?
Topoisomerase activity
Why does eukaryotic RNA polymerase require elongation factors?
To displace nucleosomes (with ATP-dependent remodeling complexes)
Transciently dislodging histone dimers
What is the corresponding segment of DNA from which a primary transcript is transcribed called?
Transcription unit
What does RNA processing require in eukaryotes?
A variety of small RNA’s of 90-300 nt (the small nuclear RNA’s, snRNA’s) and associated proteins, in addition to the enzymes involved in capping and polyadenylation
Does mRNA in bacteria undergo modification? Why or why not?
It undergoes little or no modification because it is being translated while being transcribed
What does guanylyltransferase do?
Caps the nascent mRNA chain
Note: subsequent methylation at N7 is various out in the cytoskeleton by the enzyme guanine-7-methyltransferase
What does guanine-7-methyltransferase do?
Methylated the mRNA at N7 in the cytosol
What is the purpose of capping?
Capping protects the mRNA from degradation and is needed for export out of the nucleus and for assembly of a translation complex
Are tRNAs and rRNAs capped?
They are not capped
What is AAUAAA?
The polyadenylation signal sequence which is located in the 3’ UTR and the nascent mRNA is cleaved within 10-40 bases downstream of it by a specific endonuclease.
What cleaves the mRNA after the poly-A tail?
Endonuclease
What carries out the polyadenylation reaction ?
Polyadenylate polymerase
What is the purpose of the Poly-A tail?
To stabilize the mRNA, is is needed for the nuclear export of the mRNA, and for assembly of the translation complex
What happens to the polyA tail in the cytoskeleton?
It gradually shortens
Is the Poly-A tail encoded for in the DNA? (25-200 A’s)
No
What happens after the poly-A tail is added?
Splicing
What does splicing do?
Puts together the final sequence (exons)
What enzyme carries out molecular assembly via 2 transesterications,
Splicesome
By how much can introns vary by??
Between 50 and 10k nucleotides
What do large insertions and deletions do to the splicing of an mRNA?
Large deletions or insertions do not affect splicing of the intron in most cases
All eukaryotic introns start with what and end with?
Start with GU
End with AG
What is the 5’ and 3’ splice sites called?
5’ = donor site
3’ = acceptor site
What are the two transesterifications in splicing?
- Nucleophilic attack of a branch site A on the 5’- phosphate group of the intron
- Nucleophilic attack of the now free 3’-OH group of upstream exon on the 5’-phosphate of the downstream exon
What part of the spliceosome assembly recognizes the 5’ splice site (donor site)
The U1 snRNP
What does the U2 snRNP of the spliceosome do?
Bind to the branch site (A site) recognized by U1
When U2 binds to the branch site recognized by U1, what happens?
A preasssemblex complex of U4-U5-U6 joins the others to form a complete spliceosome (inactive)
After the (inactive) spliceosome is completed, what does U5 do next?
U5 interacts with exon sequences on the 5’ splice site and the 3’ splice site and pulls the splice sites together
After U5 brings the 5’ and 3’ prime ends together, what happens next?
U6 detaches from U4 and binds to U2
Note: U1 leaves with U4
What do U6 and U2 snRNP function as? ( This occurs after U6 detaches from U4)
U6 and U2 snRNP function as ribozymes catalyzing both transesterications
How is the branch site A activated?
The RNA duplex formed with U2 forces the A nucleotide out of the RNA duplex making the 2’OH reactive
What is the UTR and what’s it do?
The untranslated regions (UTRs) can be much longer than the coding region and contains regulatory elements important for RNA stability and translation efficiency.
Note: the 5’-UTR consist of (part of) the first exon(s) and the 3’-UTR of (part of) the last exon(s)
How do variant proteins arise?
There are 3 sources for variant mRNA:
1. Alternative start sites (multiple promotors)
- Alternative splicing
- Alternative termination sites
The half life of bacterial mRNA are unstable with what half life?
What benefit does this offer?
A few minute so that bacteria can rapidly respond to changes in the environment
What does the intracellular mRNA concentration depend on?
Synthesis and degradation within the cell
What is the half life of mRNA in eukaryotes?
A few minutes for proteins involved in the cell cycle to 24 hrs for globin mRNA in erythroid cells (erythrocyte)
What is the mechanism for bacterial mRNA degradation?
An endoribonuclease makes a few cuts in the mRNA followed by 3’ to 5’ exoribonuclease action
What is the mechanism for mRNA degradation in Eukaryotic cells?
Shortening of poly A tail acts as a timer counting down the lifetime of an mRNA
Degradation occurs in specialized regions in the cytoplasm calles exosomes
What is an exosome?
A specialized region in the cytoplasm where degradation of mRNA occurs
What happens when the polyA tail becomes short than ~25 A’s?
The mRNA is rapidly degraded by either recapping followed by 5’ to 3’ degradation or 3’ to 5’ degradation
How many ribosomal RNAs are there in eukaryotes and bacteria? (rRNA)
4 in Eu
3 in bacteria
Why are rRNAs not frequently broken down?
Bases are frequently modified: methylation of bases and/or of the ribose unit as well as isomeric action of uridine to pseudouridine
What rRNA does not cluster?
5S rRNA
Where does transcription of rRNA genes and processing of eukaryotic rRNA take place?
In the nucleolus
What is the Svedberg number?
A number given to rRNA based on old techniques used to determine size
How are tRNAs made?
Derived from longer RNA precursors by enzymatic removal of nucleotides at both the 5’ and 3’ end
Both bacterial and eukaryotic tRNAs have a CCA sequence at the 3’ end. What is different about the eukaryotic tRNA?
The CCA in eukaryotes sequence is added posttranscriptionally
What s the anticodon for recognizing multiple codons for tRNA?
Inosine
How many distinct tRNAs do cell have?
40-50
Note: cancer cells have about 80-90
What do some eukaryotic tRNs contain?
An intron that has to be spliced out
What transcribe micro RNA?
miRNA is transcribed by RNA pol II that are capped, polyadenylated, processed in the nucleus, and further processed by Dicer in the cytoplasm
What is Dicer?
The enzyme that proceses ~75 nucleotides~ long genome-encoded RNAs with profound hairpin structure into double stranded silencing- RNAs (siRNAs) ! 23 nt in length
What is the RISC complex involved in?
Regulation of the stability and translatability of mRNAs
What is silencing-RNAs (siRNAs) used for?
It is incorporated into the RNA-Induced Silencing Complex (RISC)
Are all miRNAs forming siRNAs?
No, some seem to enhance/stabilize
What are the guide/scanning RNAs?
- CRISPR-RNA
2. Scan-RNA
What are some classes of long non coding RNA? (Non coding means it doesn’t make a protein)
- Telomerase RNA
- Transcriptional enchanter element RNAs
- Multifunctional IncRNAs
What are the three sources for variant mRNA?
- Alternate start site
- Alternate splicing
- Alternate termination site
