ALL THE THINGS Flashcards
What is the coding strand identical to?
mRNA strand (except T for U)
What is unique about the template strand?
complementary and antiparallel to the mRNA strand
Which strand is the template strand with respect to the DNA double helix?
the 3’-5’ strand (bottom strand)
In what direction is the mRNA strand synthesized from the template strand?
5’-3’ (but the strand is read in the 3’-5’ direction)
Are there multiple types of RNA polymerases in Bacteria?
No. Just one type.
A core RNA polymerase enzyme of how many subunits is required for nucleotide polymerization in Bacteria?
5
Can bacterial RNA polymerase proofread?
No
What does RNA poly in bacteria need in order to initiate transcription?
a promotor sequence ahead of the gene
Describe the DNA sequences that make up promotor sites in Bacterial DNA
sequences that extend 70 bp before the transcription start site to 30 bp after the start site
What confers specificity for the promoter site in Bacterial transcription?
Sigma Factor binding to the core enzyme of RNA polymerase
What is the complex of the Sigma Factor bound to the core enzyme called?
Holoenzyme
Describe the reaction that RNA polymerase catalyzes
3’-OH of the nucleotide at the end of the growing strand attacks the 5’-alpha phosphate of the incoming NTP.
What is the driving force for the reaction catalyzed by RNA polymerase?
hydrolysis of pyro-phosphate
What is the first nucleotide of the coding strand denoted as?
It is the start site and denoted as +1. Anything ahead of it is +2, etc.
What is the nucleotide preceding the start site denoted as?
-1
What are the two regions conserved in most bacterial promoter sequences?
- 35 Region
- 10 Region (Pribnow box)
What is unique about the -10 region? E.g. What is there a high concentration of?
AT content is high here
Besides binding efficiency, what are the other factors important for determining promoter strength?
consensus sequences
spacing between consensus sequences
distance of the consensus sequences from the transcription start site
What can increase the efficiency of promoters?
Negative supercoling by topoisomerases
After clearing the promoter, what happens to the RNA poly?
undergoes a major conformational chage and cannot revert
How many bp are typically found in bacterial transcription bubbles?
15-17 bp
The RNA/DNA hybrid is limited to how many bp in bacterial transcription?
7 bp
What is Rho factor involved in?
termination of bacterial transcription
What is Rho factor?
ATP-dependent helicase which can unwind RNA/DNA hybrids
Where does Rho factor bind?
to a CA-rich region in the nascent RNA chain some distance upstream of the termination site
After Rho factor binds the nascent RNA chain, what happens next?
it catches up with the RNA polymerase and dissolves the mRNA-DNA-polymerase complex
Describe Rho-factor independent termination of bacterial trancsription
termination by combined action of weak U-A bonding in the RNA/DNA hybrid and strong G-C bonding in the nascent RNA
Essentially you form hairpin or stem loop structures in both regions
What affect does RIFAMPIN have on bacterial RNA poly?
changes its conformation so that it cannot initiate RNA synthesis
Can Eukaryotic RNA poly bind RIFAMPIN?
NO! RNA synthesis is totally unaffected
What kind of DNA does Actinomycin bind to strongly?
double stranded DNA (dsDNA)
How is dsDNA affected by Actinomycin?
once bound, it is unable to be an effective template for RNA synthesis
What else can Actinomycin inhibit at high concentrations?
DNA replication
How does Actinomycin interfere with DNA? How is its action explained?
through intercalation; intercalates between two successive GC pairs
How many Eukary RNA poly’s are there?
3
Name the Eukary RNA poly’s
I
II
III
Where are the different eukary RNA polys located?
I - Nucleolus
II - Nucleoplasm
III - Nucleoplasm
What are the products transcribed RNA poly I?
18S, 5.8S, and 28S rRNA
What are the products transcribed by RNA poly II?
mRNA precursors
snRNA
miRNA
What are the products transcribed by RNA poly III?
tRNA and 5S rRNA
What affect does alpha-amantin have on the different Eukary RNA polys?
I - insensitive
II - Strongly inhibited
III - inhibited by high concentrations
How do the Eukary RNA polys differ structurally?
are differentiated by their C-terminal part; RNA pol II has long C-terminal part
What makes RNA pol II more complex than bacterial RNA poly?
12 subunits
long carboxy-terminal domain (CTD) with 52 repeats of Y-S-P-T-S-P-S
What does RNA poly II need to start?
general transcription factors
At least how many proteins are needed for eukaryotic RNA transcription?
30; functions of the proteins are comparable to Sigma factor
What are the general RNA Pol II promotor elements?
BRE - B recognition element
INR - Initiator element
DPE - Downstream promoter element
TBP - TATA binding protein
What are the general transcription factors needed for RNA pol II?
TFIIB
TBP
TFIID
TFIID
What transcription factor is associated with BRE?
TFIIB - recognizes BRE
What transcription factor is associated with TATA?
TBP - recognizes TATA box
What transcription factor is associated with INR?
TFIID
What transcription factor is associated with DPE?
TFIID
What does TFIIH do during transcription?
unwinds DNA at the transcription start point; releases RNA poly from the promoter
Also phosphorylates Ser5 of the RNA polymerase CTD
After how many nucleotides (nt) are most of the general transcription factors released?
60-70 nt
What are Elongation factors?
become associated with RNA poly so it can read through DNA associated with nucleosomes
Describe the functions conferred by Elongation factors associated with RNA poly
ATP-dependent complex remodeling that displaces the nucleosome
Histone dimer removal
What removes positive supercoils ahead of the RNA polymerase?
Topoisomerase
How is RNA poly recycled?
dephosphorylation
When you say “primary transcript,” what are you describing?
pre-RNA strand
In terms of length, what is the primary transcript equivalent to?
full length of DNA transcribed
What is the corresponding DNA segment from which the primary transcript is transcribed called?
transcription unit
What does RNA processing require?
a variety of small nuclear RNA’s (snRNAs)
enzymes involved with capping and polyadenylation
Is mRNA processed or modified in Bacteria?
No because bacterial mRNA is translated as it is being transcribed
5’- capping of the nascent RNA chain is carried out by what enzyme?
guanylyltransferase
Subsequent N7 methylation in the cytosol is carried out by what?
guanine-7-methyltransferase
What is the point of capping the mRNA?
capping protects the mRNA from degradation and is needed for export out of the nucleus and for assembly of a translation complex
What types of RNAs aren’t capped?
tRNAs and rRNAs
What is the Poly-A signal sequence identified by?
AAUAAA
Where is the Poly-A sequence located?
3’- untranslated region (UTR)
Where is the nascent mRNA cleaved at the Poly-A sequence?
10-40 bp downstream of it
What cleaves the Poly-A sequence 10-40 bp downstream from the site?
a specialized endonuclease
What enzyme carries out the poly-A reaction?
polyadenylate polymerase
Is the poly-A tail encoded for in the DNA?
No
Why even have the poly-A tail? What does it do for the mRNA?
stabilizes mRNA
needed for nuclear export
needed for assembly of translation complex
What happens to the poly-A tail in the cytosol?
it is gradually shortened
After the processing is complete, what happens to the mRNA next?
Splicing happens next
What happens during splicing of the processed mRNA?
removal of introns splices the flanking exons together into final sequence
What is a splicesome?
molecular assembly that carries out splicing
By what 2 reactions does the splicesome carry out splicing?
via 2 transesterifications
Describe the splice donor site
5’- splice site
border of 3’-end of Exon and 5’-end of Intron
starts with GU
Describe the splice acceptor site
3’- end of the intron
ends with AG
When the intron is removed, describe its structure
it resembles a lariat
What do snRNAs form when they associate with proteins?
snRNPs - small nuclear ribonucleo proteins
What do snRNPs do?
complex with other proteins to form the splicesome
base pair with the consensus sites involved in splicing and with RNA’s in other snRNPs
Which snRNPs are involved in splicing?
U1 - U6
Splicesome assembly is initiated by —
recognition of the 5’-splice site by U1 snRNP
What happens after splicesome assembly initiation?
U2 snRNP binds to the branch site
After interaction with the branch site, what forms the complete splicesome?
complex of U4-U5-U6
After construction of the complete splicesome, what happens next?
U5 interacts with exons on the 5’ and 3’ splice sites and pulls the splice sites together
After the splice sites are brought together, what happens next?
U6 detaches from U4 and binds to U2
After U6 binds to U2 what happens?
U6 and U2 snRNP function as ribozymes catalyzing both transesterifications
Does splicing require ATP? If so how many molecules are used?
Yes. 3 ATP molecules
Besides the 3’-UTR, are there any other untranslated regions in processed mRNA?
5’UTR where the cap is
What are the 3 sources of variant mRNA?
Alternative start sites (multiple promotors)
Alternative splicing
Alternative termination sites
Intracellular mRNA concentrations are dependent on what?
synthesis and degradation
Why are bacterial mRNA’s so unstable?
short half lives lasting a few minutes
allows the bacteria to rapidly respond to environmental changes
What is the half life (roughly) of mRNA in eukaryotes?
few minutes to 24 hours depending on the product
How is bacterial mRNA degradation carried out?
an ENDOribonuclease makes a few cuts in the mRNA followed by 3’ - 5’ EXOribonuclease action
In eukary mRNA degradation, what acts a timer for the lifespan of an mRNA strand?
natural shortening of the poly-A tail
When is eukary mRNA degradation initiated?
when the poly-A tail is shorter than ~25A’s
How is eukary mRNA degraded?
decapping followed by either 5’-3’ or 3’-5’ degradation
Where does eukary mRNA degradation occur?
specialized regions of the cytoplasm called exosomes
How many different rRNAs in Eukary?Prokary?
4 - Euk
3 - Pro
Where does transcription of rRNA genes and processing of rRNA take place?
in the nucleolus
How many nucleotides long is the tRNA structure?
74-95 nt
What two conformations does tRNA exist in?
clover-leaf structure
final conformation is L-shaped
tRNA’s are derived from longer RNA precursors by —
removal of nt’s at both 5’ and 3’ end
What do some eukary tRNAs contain that needs to spliced out?
an intron
Both prokary and eukary tRNAs have —
a CCA sequence at the 3’-end
How is the CCA sequence in eukary tRNA different from its prokary counterpart?
CCA sequence is added post-transcriptionally in eukarys
What do modified bases in tRNA allow?
better recognition when binding
Inosine (modified adenine) allows for recognizing of multiple codons
By what ribozyme are eukary tRNAs processed?
RNaseP
Our genome contains how many miRNAs?
400
What are miRNAs transcribed by?
RNA Pol II
What caps, poly-adenylates, and processes miRNA?
Dicer
Dicer processes 75 nt long genome encoded RNAs with profound hairpin structures into what?
double stranded silencing RNAs (siRNAs)
One of the strands of siRNA is incorporated into what?
RNA-induced silencing complex (RISC)
What is the RISC complex involved in?
regulation of the stability and translatability of mRNA’s
