Genetic information - Translation Flashcards
what determines the rate of translation
initiation
-its the slowest step
what tRNA does bacterial mRNA bind to
formylmethionine-tRNAf
what is the function of each initiation factor
IF1 - binds in A-site, prevents elongator tRNA from binding
IF2 - binds the GTP and fMet-tRNAf
IF3 - prevents association with the 50s subunit, ensure fidelity of the initiation codon selection
forms the initiation complex
what do prokaryotic mRNA possess to help locate the initiation codon
a Shine-Dalgarno sequence that base pairs to the 3’ end of the 16s rRNA
where is the start codon (AUG) placed
ribosome P-site
about 10 bases 3’ of the Shine-Dalgarno sequence
what happens after the start codon is placed
the 50s subunit binds and IF1/3 dissociate
what is eIF3
eukaryotic equivalent to IF3
binds eIF4G and the 40s subunit
outline eukaryotic initiation
(long essay awnser)
eIF2 + the small 40S ribosome subunit + methionyl-tRNAimet + GTP bind to each other
The 40S subunit binds to mRNA with other initiation factors (eIFs) bound to the cap and poly A tail regions
The 40S ribosome then “scans” the mRNA looking for the AUG initiation codon: usually uses first AUG it encounters
More efficient if this AUG is within the Kozak Consensus
eIFs then dissociate and the 60S subunit binds
Intact cap and tail regions are essential for initiation
what are the similarities of initiation between eukaryotes and prokaryotes
eIF2 bound to GTP and brings in first tRNA
eIF3 and eIF1 ensure accuracy of initiation and prevent association of 60S large subunit
why does 4E-BP’s bind to eIF4E
4E-BP has a very similar sequence motif to eIF4E binding site of eIF4G
why does does 4E-BP dissociate with eIF4E
4E-BP gets hyperphosphorylated
structure of eIF2 and what part binds GTP where specifically is it phosphorylated
alpha, beta and gamma subunit
gamma
Ser 51 on alpha subunit
what are the implications for elF2 when it is phosphorylated
it cannot bind GTP
in terms of elF2, outline what happens from the point of stress
1 - stress
2 - increase in elF2 kinase
3 - increase in elF2-α phosphorylation
4 - general translation is blocked
5 - paradoxical increase in translation of specific mRNA
what do viral-infected cells produce and what does that induce and what does it cause
interferons (IFN)
induces protein kinase R (PKR) in neighbouring cells - anti-viral state
causes reduction in protein synthesis in both host and viral mRNA - prevents further infection
function of chaperones
heat shock proteins
prevent illicit liaisons between proteins
(interactions between exposed hycrophobic regions)
what keep PERK inactive
BiP binds to PERK and keeps it in an inactive monomeric state
how is PERK activated
BiP dissociates to bind to unfolded proteins
activating PERK dimers
what is Walcott-Rallison’s disease
recessive
Loss of PERK function – patients develop Type I diabetes, growth retardation, multiple other effects
in terms of a modified base in tRNAPhe, what is the function of pseudouridine
has more possibilities for H-bonding (a,d), and more rigidity
Better for stability in tertiary structure and interaction with ribosome
what is step 1 of the amino acid activation process
Amino acid + ATP + enzyme ==
= enzyme-AMP-(bond)-amino acid + PPi
step 2 of the amino acid activation process
enzyme-AMP—amino acid + tRNA =
= Aminoacyl—tRNA + AMP + enzyme
how much ATP is used during the 2 step process of aa activation and what is their purpose
2 ATP is used
the second is used to drive the reaction
what happens if an aa acid that is too large tries binding with an incorrect aminoacyl-tRNA transferase
if its too large it wont be able to enter the acylation active site
what happens if an aa thats too small tries binding with an incorrect aminoacyl-tRNA transferase
it can enter the acylation active site but it also enters the hydrolytic active site
so it gets hydrolysed
how does the corresponding aa bind to its aminoacyl-tRNA transferase
enters acylation site but cannot enter the hydrolytic site
properly acylated tRNA
properly charged tRNA
what plays no role in loading of the correct amino acid onto the tRNA
the anti-codon
what does tRNA wobble usually involve
nucleotides U or inosine (I) at the first position of the anti-codon
what is inosine
made by deamination of adenine
what can I base pair with
A, U or C using 2H bonds
what are the key features of bacterial 50s ribosome subunit
23S and 5S rRNAs + proteins L1– L31
23S rRNA forms 6 domains (I-VI)
Contains the peptidyl transferase centre (PTC)
Contains the peptide exit tunnel
what are the key features of the 30s ribosomal subunit of bacteria
16S rRNA + proteins S1- S19
Contains the “decoding centre” (DC)
Helix 44 of 16S rRNA forms the A and P tRNA binding sites
3’ of 16S rRNA complements the Shine-Dalgarno
what is a ribozyme
RNA thats capable of acting as an enzyme
what is the evidence to suggest that bacterial ribosomes are ribozymes
No ribosomal proteins located in the PTC (peptidyl-transferase centre) – nearest ones are 15-18Å away – too far to take part
Ribosomes that are largely depleted of protein still have peptidyl-transferase activity
how is the ester bond broken between amino acid and tRNA
nucleophilic attack by amino group of the incoming aminoacyl-tRNA
what mutation causes a decrease in the rate of peptide-bond formation
Mutation of 2’-OH of A76 of peptidyl-tRNA decreases the rate of peptide bond formation ~million-fold
what mutations do not effect the rate peptidyl-transferase reaction and what does this mean
mutations of rRNA bases
Therefore ribosome helps to position substrates and water molecules which aids proton transfer and stabilisation of intermediates - basically decreasing necessary activation energy
what is the speed and error rate at which ribosomes work
10-40aa per second
1 in 10,000 errors
why is tRNA always base paired with mRNA
prevents frameshifting
ensures accuracy
what is trigger factor (TF)
coordinates folding/elongation rates
purposes of the exit tunnel
Nascent chain passes through the ribosome exit tunnel
Protects new chain from inappropriate interactions
Allows it to sample multiple conformations
function of chloramphenicol
blocks peptidyl transferase
function of erthromycin
blocks elongation by binding to the 23S rRNA tunnel
function of tetracycline
prevents amino acyl tRNA binding
name two aminoglycoside antibiotics and their function
paromomycin
streptomycin
bind to decoding centre (DC) and introduce errors
what is puromycin and its functions
is an analogue of tyrosyl aminoacyl tRNAtyr and causes premature peptide chain termination - also inhibits protein synthesis in eukaryotes
Can use puromycin analogues as tags to assay protein synthesis
what recycles ribosomal subunits
ABCE1
purpose of mRNA surveillance in terms of nonsense-mediated decay (NMD)
mechanism used to detect premature termination codons (PTC) before translation
prevents accumulation of truncated proteins
what happens to the pioneer round of translation when it binds to a mRNA with a PTC
ribosome does not reach the EJC and cannot displace it
where are EJC’s placed on mRNA
20-24 nucleotides upstream of each splice site
what is considered a PTC
Stop codons more than 50-55 nucleotides upstream from most 3’ exon junctions are recognised as premature.
what is recruited to a mRNA with a PTC
SURF
what does SURF contain
SMG1 (kinase)
UPF1 (RNA and ATP-dependant helicase)
eRFs (translation termination factors)
what brings SURF to the PTC containing mRNA
UPF1 interacts with UPF2, bridging SURF complex to EJC
function of SMG1
Following SURF binding, SMG1 phosphorylates UPF1 and causes dissociation of eukaryotic Release Factors and ribosome.
what does phosphorylated UPF1 cause
recruits SMG7
decays enzymes
what does a SURF + EJC create and cause
DECID complex
causes aberrant termination
regarding no-go decay (NGD) what happens if the ribosome doesn’t dissociate
the reading frame can shift to the +1 frame and then make an aberrant protein
