Mitchell - Molecular Basis of Gene Expression Flashcards
which interactions stabilise RNA structure?
base pairing (H bonds) hydrophobic base stacking interactions
what secondary structure does RNA usually form?
short irregular stem loops
which metal ion is often attached to RNA to stabilise it?
Mg2+
compare the major/minor grooves of DNA and RNA
RNA major groove is deeper and narrower
RNA minor groove is shallower and broader
which protein is associated with cellular RNA?
ribonucleoprotein particles
give the carbon positions where base pairing interactions occur on pyrimidines and purines
(2) 3 and 4 pyrimidines
(2) 1 and 6 purines
give the base pairs involved in the wobble and imino interactions
U-G wobble
G-A imino
describe base triples and tetraloop RNAs
base triples are 3 bases bonded - increases base stacking interactions (more stable)
tetraloops RNA are 4 nucleotide long loops - smallest possible loops (compact)
name 4 tertiary structures
long-range base-pairing/coaxial stacking of helices/A-minor motif/pseudoknot
which is the most abundant cellular RNA?
rRNA
name the 2 enzymes that process precursor RNAs into cellular RNAs
endo/exo ribonucleases
what do endo and exo ribonucleases do?
exo: 5’ to 3’ or 3’ to 5’ - degrade RNA from the end
endo: cleave specific structures/sequences from RNA
what functional group always ends the 3’ end and the 5’ end?
3’ - OH
5’ - triple phosphate
give the standard equation for the hydrolysis of pyrophosphate
(n)RNA + NTP = (n+1)RNA+PPi
PPi + H2O = 2Pi
what is the location of the E.coli promoter region?
-35 and -10 nucleotides from transc. start site
name and describe the function of the 5 subunits of RNA polymerase
2x beta: forms claw that binds DNA and has catalytic activity
2x alpha: provides contacts with protein factors that regulate transc, activity
omega: required for assembly and stability of core enzymes
how was it proven the sigma factor binds tightly to RNA pol?
2 tubes:
1 tube: RNAP holoenzyme + radioactively labelled DNA and then unlabelled DNA added
2nd tube: RNAP (no sigma factor) + radioactively labelled DNA and then unlabelled DNA added
what happened: when labelled DNA dissociates from RNAP unlabelled DNA takes its place and labelled DNA enters filtrate
in tube with no sigma factor the radioactivity in the filtrate increased more quickly, shows greater dissociation of DNA from RNAP with no sigma factor (as it binds less tightly)
what does the sigma factor do?
recognises the -35 and -10 promoter sequence
how is transcription initiated in prokaryotes?
RNA pol holoenzyme (RNA pol and sigma factor) bind DNA at promoter
after unwinding and the production of a small piece of RNA the sigma factor is lost and NusA binds
what does NusA do?
Prevents stalling of the polymerase and premature
transcription termination
name the 3 methods of intrinsic transcription termination
- RNA stem-loop structure
- GC rich region at stem base
- 3’ U rich tail
RNA pol pauses at the hairpin structure and the (RNA)U and (DNA)A pairing causes destabilisation
which RNAs do pol’s 1, 2 and 3 act on?
1: 18s, 25s, 5.8s rRNA
2: mRNA small stable RNAs
3: 5s rRNA and tRNA
what feature is present in eukaryotic pol 2
extended c-terminal domain
which transcription factor (TF) is required for pol II? what does this achieve?
‘general TFs’
facilitates RNA pol turning into preinitiation complex
what do ‘specific TFs’ do?
influence transcription of particular genes
describe the interactions between the TATA box and the TBP
TBP in TFIID recognises TATA box
TFIID and TBP bind to TATA
DNA bends and minor groove increases allowing other TFII’s to bind
define and explain the ‘torpedo’ method of transcription termination
it is pol II termination coupled with mRNA 3’ processing
the transcript is cleaved by cleavage/phosphoadenylation complex
cleavage allows degradation of downstream fragment by exonuclease Xrn2
(Xrn2 binds to unprotected 5’ end and displaces pol II from the DNA)
Pol III terminates transcription at stretches of A bases in the template seq
name 3 methods of eukaryotic mRNA processing
5’ end being capped
introns removed via splicing (pre-mRNA splicing by spliceosome)
polyadenylation of 3’ end
what strucutral charecteristics of eukaryotic mRNA protect it from exonucleases?
The 5’ 7G cap structure and non coded poly A tail
where is the open reading frame situated?
between the non coding 5’ leader sequence and 3’ untranslated region
what structure is involved in the 5’ cap?
5’ to 5’ triphosphate linkage. The guanosine residue is universally methylated at the N7 position
what 3’ end processing does eukaryotic mRNA undergo
the 3’ end is formed by a coupled cleavage and polyadenylation which recognises the AAUAAA sequence. Polyadenyl polymerase adenylates the 3’ end.
what do the splice site consensus sequences recognise?
the 5’, the branch point, and the 3’
what is the spliceosome made up of?
5 RNPs involving the small nuclear RNAs (snurps)
what is the catalytic mechanism of splicing?
involves 2 transesterification reactions instead of 1 ligase reaction.
R-OH + R’-O-R’’ = R-O-R’’ + R’-OH
2’ hydroxyl group at the branch point adenosine attacks the 3’ phosphate of the 5’ exon.
5’ to 2’ phosphodiester bond gives a looped lariat.
The generated 3’ hydroxyl group attacks the 5’ phosphate of the 3’ exon releasing the lariat.
name the initiation codon and its base sequence
methionine AUG
how is the shine dalgarno sequence recognised?
by 3’ end 16s rRNA
which bases can bind with inosine?
A C U
give the rRNA that makes up the small and large subunit of the prokaryotic ribosome
small subunit: 16s
large subunit: 23s 5s
give the rRNA that makes up the small and large subunit of the human ribosome
small subunit: 18s
large subunit: 5.8s 28s
name the elongation factors involved in eukaryotic and prokaryotic translation
euk: EF1A brings aa-tRNA into A site
EF2 translocates peptide bond
prok: EF-Tu brings aa-tRNA into A site
EFG translocates peptide bond
how many GTP are hydrolysed in a/a ribosome incorportation?
2
give and explain the name of both prok and euk initiator tRNAs
prok: tRNA Met f (methionyl group formylated)
euk: tRNA Met i (methionyl group not modified)
describe translation initiation in e. coli
tRNA met f binds P site (by IF2) forming ternary complex of GTP+IF2+tRNAmetf
30s associates with mRNA and ternary complex which associates with 50s subunit
GTP hydrolysis by IF2 releases IF2 and forms preinitation complex
describe translation initiation in euks
ternary complex (tRNAmeti+GTP+eIF2) binds 40s
complex interacts with 5’ cap by cap binding complex eIF4F
eIF4F scans mRNA until recognition initiation complex
scanning property of eIF4F driven by helicase activity
when tRNA is positioned at initiation codon eIF2 and GDP are released (requires hydrolysis of GTP)
binding to 60s requires eIF5 until the recognition of the Kozak seq (AccAUGG)
describe translation termination (same for prok and euk)
termination codons recognised by release factors
RF1/RF2 (eRF1) binding at A site releases polypep via hydrolysis
RF3 (eRF3) binds RF1 (eRF1) after polypep release
RF3 (eRF3) hydrolyses GTP to release RF1/eRF1 from ribosome
ribosome separated into subunits by EFG
what do many widely used antibiotics target? name 2 that do this
prokaryotic ribosome
chloramphenicol, erythromycin
how does diphtheria toxin act as a ribotoxin?
targets eukaryotic ribosomes (therefore ribotoxin)
diphtheria toxin transfers an adenosine ribosyl residue from NAD+ to a modified histidine residue in EF2 –> inactivated EF2
which part of the ribosome do toxins ricin and sarcin target?
EF2 interacts with the ribosome through a specific feature of the 23s rRNA called the ricin/sarcin loop (this is what the toxins target)
how many ribosomes can ricin inactivate per minute
~1500
