Translation (Cundliffe) Flashcards
(166 cards)
1
Q
What is gene expression?
A
DNA –> mRNA –> Protein
DNA is transcription
mRNA and Protein are translation
2
Q
Where does the single translation start site begin?
A
On the start of the open reading frame
3
Q
Where does the ribosome bind?
A
At the cap of the UTR
Makes eukaryotic translation different compared to prokaryotic translation
NO SHINE-DALGANO RECOGNITION OCCURING
4
Q
What does the ribosome do at cap?
A
Consumes ATP to provide energy for the scanning process
5
Q
How many ORF do bacteria have?
A
3 (ORF 1, 2, 3)
In between ORFs, there are multiple translation start sites
6
Q
What are polycistronic mRNAs?
A
Polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs
7
Q
Where does all protein synthesis take place?
A
On polyribosomes
8
Q
What is the template strand?
A
3’ to 5’
9
Q
What is the initial start amino acid in translation?
A
Methionine
Is at the N terminus (amino)
10
Q
What are the start codons for translation?
A
AUG and GUG
In bacteria, around a 1/4 of all reading frames start with GUG
11
Q
What happens when you add a ribose to N1 on U base?
A
You get the normal U side, uridine
Present in all RNA molecules
12
Q
How is uridine generated in a RNA molecule?
A
Psuedo-U-Synthase rotates the base so C5 is linked to the base and NOT N1
This novel bond defines pseudo U
13
Q
What is another form of modification?
A
Reduction
dihydrouracil (UH2 or DHU) is formed from uracil
DOESNT FORM A FLAT RING, IS INSTEAD CROOKED
14
Q
How is hypoxanthine generated?
A
Post transcriptionally in RNA strands by the deamination by specific residues
15
Q
What were the first tRNA’s that were crystallised?
A
Yeast tRNA(phe) Yeast trNA (ala)
16
Q
How do you get from the secondary structure to the tertiary structure?
A
By a process called co-axial stacking
17
Q
How are unpaired interactions in the T-loop and D-loop formed?
A
Every nucleotide in a tRNA that isn’t paired in secondary structure is paired in the tertiary structure
18
Q
Where is there Watson-Crick pairing in the codon-anticodon interaction?
A
On the 1st and 2nd mRNA and the 2nd and 3rd tRNA
19
Q
Where is the wobble position on the codon-anticodon interaction?
A
On the 3rd mRNA and 1st tRNA interaction
20
Q
What was at the first position of the anticodon?
A
I was at first position of the anticodon
21
Q
What can tRNA’s do?
A
They can recognise multiple codons (1,2,or 3)
Codons can be recognised by multiple tRNA’s
22
Q
Why doesn’t it take 61 tRNAs to recognise 61 sense codons?
A
Due to wobble pairing
23
Q
What is wobble pairing?
A
A wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules
Wobble pairs decode U3 and C3 codons
24
Q
What is tRNA sparing strategy for bacteria?
A
They omit A34 and G34 steps in and replaces where required
Happens in 7 cases
16 tRNAs are spared out of 32
Bacterias use 16 G34 tRNAs, they use 45 tRNA’s needed to decode 61 sense codons
25
What is tRNA sparing strategy for eukaryotes?
We also omit A34 or G34
We deaminate A34 and turn it into an I34
Eukaryotes use 8 I34 plus G34 tRNAs EXCEPT YEAST
26
How are amino acids attached to tRNA's?
By aminoacyl - tRNA synthases
27
What is the equation for activation of amino acids?
aa + ATP + enzyme [aa-AMP-E] + PPi
28
What must aaRS enzymes select?
1 amino acid from 20
1-5 tRNAs from 40-50
29
Is GlnRS activation or domain?
activation
30
Is ThrRS activation or domain?
domain
31
What is the first step of isoleucine activation?
1. Cognate activation
32
What is the second step of isoleucine activation?
2. Cognate charging
tRNA that is cognate for isoleucine comes in and picks up isoleucine that is cognate for adenylate
33
How does valine become activated?
By the enzyme that is cognative for leucine
The tRNA that is cognative for isolecuine comes in a picks up whatever amino acid that is activated, so false charge intermediate is formed
34
What are the two possibilities from an intermediate charge?
1) Nothing happens to it, the false product dissociates from the product (DONT WANT)
2) Hydrolytic editing, becomes cleaved, amino acid becomes chopped out
35
What is the rate of false activation if you give the enzyme the same amount of valine and isoleucine?
1 in 200
36
What does tRNA do?
It picks up the false activated intermediate with 100% effiency
37
What is the efficiency of hydrolytic editing?
799 in 800
38
What is the efficiency of false charged intermediates that survive hydrolytic editing?
1 in 800
39
What is the total error frequency?
1 in 200 x 1 in 800 = 1 in 160000
40
In an aaRS enzymes, where is the amino acid added?
On the activation site
| Thumb of the tRNA
41
What happens if the amino acids gets into the editing site?
It will get chopped out
42
What does Tu-GDP + Pi do?
It carries aminoacyl tRNA into the ribosome
43
What does the NMR experiment tell us?
That the protein shape changes depending on whether GDP or GTP is present
High affinity when GTP is bound
Low affinity when GDP is bound, how it gets out
44
What is the role of elongation factor TS?
EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu (elongation factor thermo unstable), catalyzing the release of guanosine diphosphate from EF-Tu. This enables EF-Tu to bind to a new guanosine triphosphate molecule, release EF-Ts, and go on to catalyze another aminoacyl tRNA addition.
45
What are the elongation factors in bacteria?
- EF-Tu
| - EF -Ts
46
What are the elongation factors in eukaryotes?
- eEF- 1A
| - eEF - 1B
47
What happens if the ternary complex is cognate?
aa-tRNA retained in A site
48
What happens if the ternary complex is NOT cognate?
aa-tRNA ejected from ribosome
49
How do we know if ribosomes are proofreaded?
If the number is greater than 1
50
What has the ribosome evolved to do?
To deliver optimal growth rate
Also need versus cost of accuracy
51
What do ribosomes mutations affect?
Fidelity
Ribosomes must do more than generating a water free zone to enable codon-anticodon pairs
Monitor codon-anticodon pairs require decoding site
52
What is bacterial ribosomes structure?
70S 50S + 30S
Roughly 50 proteins in bacteria
53
What is eukaryotic ribosome structure?
80S 60S + 40S
Roughly 80 in eukaryotes
54
How many protein-protein interactions are there in the smaller ribosomal subunit?
None
55
Where is the S12 placed in 30S subunit?
In the decoding box
56
What occurs in the decoding box in the 30S subunit?
Codon-anticodon interaction in ribosomes
57
Where is the peptidyl transferase centre on 50S subunit?
In the centre of the 50S
It is the peptide bond forming site of the ribosomes, no proteins near it, rougly 18A away
58
Where are peptides made?
In the peptide tunnel in the 50S subunit
59
How is a ribosome prep made?
Through a 'dirty' prep, one that hasn't been washed via centrifugation
You get a protein by washing them off the ribosomes, quick way of getting several of the enzymes
60
Where is the tRNA NOT present at any given time?
On the E and A site simultaneously
61
What is the rate limiting step?
The rate determining step is the slowest step of a chemical reaction that determines the speed (rate) at which the overall reaction proceeds
62
What is the 2 functions of translocation?
1. Free up the A site and ready to receive the next aminoacyl-tRNA
2. Move the ribosome along the mRNA so when the A site is free, it will have the next mRNA in it in the ribosome
63
What does mRNA do in translocation?
It moves from outside of the ribosome into the A site of the ribosome and when that happens the tRNA move together. Red tRNA has moved from A to P and Blue has moved from P to E
64
How do tRNAs move?
tRNAs go via hybrid site (P/E) & (A/P)
65
In hybrid sites, what are the two phases?
1) First phase, spontaneous
enters the A/A site and pushes blue tRNA into P/P site
2) Second phase, requires EF-G + GTP
66
What does EF-G do to tRNA?
It pushes tRNA from 30S A to 30S P site
67
Which 2 elongation factors occupy the same site during the elongation factor cycle?
1) EF-Tu
| 2) EF-G
68
What are release factors?
A release factor is a protein that allows for the termination of translation by recognizing the termination codon or stop codon in an mRNA sequence
Bacteria = RF1 (UUA and UAG) and RF2 (UAA and UGA)
Eukaryotes = RF3, provides energy for GTPase for this purpose
69
What does release factor do?
It triggers peptide release via peptidyl transferase
Causes peptidyl transferase onto water instead of transferring it on to amino acid attached to tRNA on the A site
70
What is present at the top of ERF?
GGQ box
Glycine, Glycine, Glutamine box
71
What does ribosome release factor (RRF) do?
Along with EF-G it generates free 70S
Cleans out the ribosome, ribosome dissociates into two subunits
72
How many tRNAs to E.Coli have?
1) ordinary tRNA
| 2) initator tRNA (tRNAf) used at AUG or GUG
73
Which enzymes discriminates between the 2 tRNAs?
Transformylase
74
During the initiation process, which site does not process?
The E site
75
What are the difference from bacterial translational initiation?
- No shine-Dalgarno recognition
- 40S binds to mRNA-CAP
- occurs by protein-protein interactions
- complexity and large numbers of eIFs
76
At the 5' end (CAP), what is bizarre about the sequence?
- GTP methylated on the 7th position is added on the reverse polarity
- 5' triphosphate linkage
- What supposed to be a 5' mRNA, you actually have a 3' OH group
77
How many residues to A residues does the tail have?
200
78
What is responsible for the circular nature of eukaryotic mRNAs?
The tail
Attached to the tail of mRNAs there are poly-A binding proteins
Often more than 1
Protein attached to the tail interact with proteins that interact with the cap
79
What eIFs bind to the small subunit of ribosomes?
eIF1, eIF1A, eIF3, eIF5
80
What eiF binds to the large subunit of ribosomes?
eIF6
81
What does eIF2 bind to?
met-tRNAi
82
What does eIF4 group bind to?
mRNA CAP
83
What do eIF5 and eIF5B do?
They release other eIFs
To clean out other factors that have done their job
84
How many genes are the products of eIFs?
30 genes
85
What is the role of the 43S pre-initiation complex?
The 43S preinitiation complex (43S PIC) is a ribonucleoprotein complex that exists during an early step of eukaryotic translation initiation.
PIC binds to the cap of the mRNA, then scans, until it gets to the coding sequence. PIC breaks down as prelude to initiation of translation
PIC binds to the cap with the eIF4 bound to it
86
What happens whilst the PIC is forming?
eIF4 binds to the cap of the mRNA
Is an ATP consuming process
87
What is keeping this whole process bound to each other?
The eIF3 and eIF4 bound to the complex on the cap
88
What does the PIC do to the mRNA?
The PIC migrates and drags the CAP with it so the mRNA loops out
89
What does eIF1 do in the scanning complex?
It keeps 43S 'OPEN' during scanning
Factor 1 leaves after scanning
Factor 5 moves into the position factor 1 was sitting in
It triggers the GTPase activity of Factor 2
90
What does eIF4A do in the scanning complex?
RNA helicase
Energy that comes for scanning comes from eIF4A
Also unfolds RNA structure
91
What gets removed after eIF5 replaces eIF1 after the scanning process?
The GDP + Pi on eiF2 gets removed due to GTPase activity
92
What happens when the 43S complex closes?
AUG gets recognised
48S initiation complex begins (TRANSLATION MODE)
93
What does eIF5B do?
It brings in GTP and hydrolyses it
To also get the large subunit (60S) bound into it
94
What removes eIF3 to allow for the 80S initiation complex?
eIF5B clears out remaining factors and allows 60S to join
Once eIF3 comes off the 40S ribosomal subunit, the cap is no longer tethered to the ribosomal subunit
95
What does eIF2B do?
To come in and displace GDP to give a protein-protein dimer, from which GTP displaces 2B
96
What is the only cap binding protein?
4E
97
What is protein 4G?
It is a scaffold protein
It is bound to eIF3
It is attached to the cap by 4E
Has multiple binding sites for 4A
98
How does the scanning process stop?
Until it finds the initiation codon in Kozak consensus sequence
99
What is the Kozak consensus sequence?
The Kozak consensus sequence is a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG. The Kozak consensus sequence plays a major role in the initiation of the translation process
100
What are leaky mutations?
Some amino-acyl tRNA recognises the stop codon and translation continues
Leakiness is accidental
Just how leaky varies on - which stop codon were thinking of
- which gene were thinking of
- where the mutations occurs in the gene
101
How to plants replicate their RNA?
The eznyme that needs to replicate their RNA is NOT present inside the plant
There is no RNA-directed polymerase in plant cells
Virus must encode its own replicase
102
What are the two products seen from the replicase gene?
1) Big protein 1000 amino acids in length
| 2) Another protein product from the same gene (LEAKY UAG, Tyrosine inserted)
103
What is the gene for release factor 2 (RF2)?
pfrB
Has 397aa in length
104
What does pep-tRNA do?
It slips CUU to UUU
Causes a +1 frameshift
105
What is a slippery sequence in dnaX gene E. coli?
A AAA AAG
Ribosomes slip when they come onto this sequence
106
What is a -1 frameshift?
tRNAs slip
AAG to AAA
AAA to AAA
107
How many subunits does DNA pol III have?
2
Tal and Gamma
71 KDa 47KDa
Equimolar amounts
108
What is reverse transcription?
ssRNA --> DNA-RNA --> dsDNA
You need an enzyme called RNA dependent DNA polymerase to do this type of reaction
Virus has to produce it
109
What do retroviruses need?
Reverse transcriptase (RT)
110
How many active sites does reverse transcriptase have?
2 active sites
111
What gets translated?
The newly synthesised retroviral mRNA?
112
What does the retroviral mRNA code for?
gag and pol
113
What are gag proteins?
They are structural proteins
There are 3 of them in the gag genomic sequence
Gets translated in the form of polyproteins
114
What is pol proteins?
They are enzymes including RT
Also intergrase and protease
Gets translated in the form of polyproteins
pol has no start
115
How big is the gag-pol overlap?
200nts
116
How are RT's made?
As part of a Gag-Pol fusion
Switch from gag to pol requires a -1 frameshift and without it, the virus cannot replicate
117
How do leukaemia viruses generate their gag-pol polyproteins?
Through stop codon readthrough
118
What virus gets reprogrammed through a -1 frameshift?
HIV-1
119
What do you get out of 1% of the time in lac Z gene?
A real normal lac Z protein with no inserts in it
It gets past the STOP codon without translating anything
+6 hop
CUU --> CUA
leu leu
120
TRUE OR FALSE: Can hops move in and out the same reading frame?
True
121
What is T4 phage gene 60?
It uses and needs a topoisomerase thats encoded by gene 60 by T4 phage
122
How much % of efficiency did John Atkins say?
60%
Without 60% efficiency, the phage could not replicate
123
What is the name of the active site that makes peptides?
Peptidyl Transferase Centre
124
What does Puromycin resemble?
It resembles the 3' end of aa-tRNA
3' end --> Where the amino acid is added
Puromycin is an acceptor substrate for peptidyl transferase
125
What happens to the peptidyl puromycin in the puromycin reaction?
It falls off the A site
126
What work did Robin Monro do to prove the reaction was catalysed in the 50S ribosome?
Monro's ‘fragment reaction’, the ribosome catalyzed reaction of a fragment of formylmethionyl-tRNA with puromycin
127
How is the fragment reaction sustained?
By the larger bacterial or eukaryotic subunit
128
What do donor substrates require?
Donors require amino acids with blocked amino groups
If you block the amino group, you get a positive charge
Can block it with either N-acetyl or N-formyl
Donor substrates also need 2'-OH
129
How is peptidyl and amino tRNA held in optimum position for peptide bond formation?
With hydrogen bonding interactions from residues from rRNA
Also positioned with one or more residues from the 23S RNA (A and P loop)
130
What does the A and P loop do?
They stabilises the positioning of the bonding of tRNA
131
Where is the GTPase activation centre located?
On the large ribosomal subunit
132
What is the role of the GTP in ribosome mRNA?
To get rid of the Tu
133
How do you get rid of the protein after its done its job in the ribosome?
By getting it to hydrolyse GTP
The protein changes its conformational state and loses its affinity to the ribosome and it comes out
134
How would you try to avoid GTP from being hydrolysed?
By using GMP
The oxygen bond in the middle avoids hydrolysation
135
What is a GTP box?
When GTP is going to get hydrolysed, one or more components have got to be put into the box in order for hydrolysis to occur
136
What does Isoleucine 60 have?
A flexible loop called switch I
That can move and the gate can open
137
What does Histadine 84 have?
A flexible loop called switch II
138
What was H84 found to do?
H84 acts as a base to activate catalytic H2O by capturing a proton
139
TRUE OR FALSE? TC can bind into A/A site
False
140
How many dimers are present in a ribosome?
2 dimers that form a store that the ternary complex first meets on its way into the ribosome
Also where protein TU binds to
141
What is the SR loop of the 23S RNA?
Small very conserved loop of the ribosomal RNA
This is not a binding target, but importantly, its nearby in GAC
142
What does the L12 stalk bind to?
It binds to Tu
143
What does the L11 + "L11-23S RNA" bind to?
It binds to the tRNA elbow
144
Where does the Ternary Complex bind onto?
Onto the L12 stalk of the GAC
145
In EF-Tu:GTP, where is H84 positioned?
H84 gets repositioned by SR loop to activate H2O
146
What happens after GTP gets hydrolysed?
Pi leaves
If Pi doesn't leave, the protein will not notice if the bond hasn't left
147
How does GTP move back to GDP?
- A movement of Switch 1
- Histadine backs off
- The GTP box becomes a GDP box
148
What happens after Tu-GDP departs?
tRNA rotates A/T to A/A
149
What is the signal for GTPase activation?
The ribosomes nods to the left in acceptance in cognate for tRNA
Ribosome also sticks up its shoulder
The ternary complex squeezes
150
What 3 things does GTPase activation involve?
1. aa-3'-tRNA acceptor stem released from EF-Tu
2. GTP box opens
3. SRL positions H84 for catalysis
151
What 3 things happens after GTPase (Pi release)?
1. GTP box disintegrates, switch I moves away from GDP and H84 moves back towards SRL
2. Domain I moves and rotates back to the GDP associated position
3. Tu-GDP leaves the ribosome
152
What 3 things consist of the ternary complex?
1. aa-tRNA
2. EF-Tu
3. GTP
153
In the EPA site, what must tRNA do?
It must rotate or it falls out
Rotation is called accommodation
Rotation is in-excess of 90*
154
Where does the energy for accommodation come from?
From the relaxation of tRNA into the A/A site
155
How many helical stems do 16S rRNA have when folded into its natural (secondary) structure?
46 helical stems
156
Which ribosome is recognised mostly with codon-anticodon recognition?
Ribosome S12
157
What does 16S RNA A1493 assess?
Codon pairing 1
| Anticodon pairing 3
158
What is near cognate?
Closely similar
| Fits in lock but won't turn
159
What is non-cognate?
Not closely similar
| Won't fit lock
160
What is cognate?
Correct key
161
What happens if a non-cognate ternary complex comes in?
It gets rejected
There's no GTPase
G530 cannot flip out
162
What happens if a near-cognate ternary complex comes in?
They can get past selection 1 and G530 bases flip out
The tRNA gets retained
Signal from decoding site triggers GTPase and Tu-GDP departs
163
What happens to the aa-tRNA after the GDP has departed?
It can become cognate or near-cognate
164
What does a cognate aa-tRNA look like?
If it cognates, it accommodates and aa-tRNA rotates into A/A site
165
What does a near-cognate aa-tRNA look like?
tRNA gets rejected
166
What is the acceptable error frequency for bacterial ribosomes?
1 in 10^4
