Translation Flashcards
1
Q
What are mRNA sequences read by?
A
ribosomes
2
Q
What is a codon?
A
unpunctuated base triplets (that encode a specific amino acid)
3
Q
How many codons are there?
A
64
4
Q
What is degeneracy?
A
some amino acids have more than one codon
5
Q
What is the special function of AUG?
A
only codon for methionine and is used as the initiation codon (tells ribosome which reading frame to use)
6
Q
How many codons encode proteins?
A
61
7
Q
What do the other 3 codons do?
A
stop codons (they do not encode amino acids)
8
Q
How many standard amino acids are there?
A
20 (obviously they have more than one codon)
9
Q
Do codons overlap in the RNA?
A
no they do not overlap and there are no spaces
10
Q
What are the 3 stop codons and what do they do?
A
UAA: You are angry
UGA: You go away
UAG: You are gone
instruct the ribosome to stop translation
11
Q
What is the first codon to initiate protein synthesis?
A
AUG
12
Q
What are the codons that we need to know? (8)
A
AUG UUU, CCC, GGG AAA UGA UAG UAA
13
Q
What is the open reading frame or ORF?
A
the in-frame sequence between the initiation codon and the termination codon
14
Q
The 5’ end of the mRNA corresponds with which part of the protein?
A
the N-terminal
15
Q
The 3’ end of the mRNA corresponds with which part of the protein?
A
C-terminal
16
Q
What is the name for the 5’end?
A
amino terminus
17
Q
What is the name for the 3’ end?
A
carboxy terminus
18
Q
What did Dintzis do in 1961?
A
(this is how they found out which direction the protein was being made) used radioisotopes to follow globin synthesis in reticulocytes (immature erythrocytes)
19
Q
Can RNA interact directly with amino acids to specify their order?
A
no
20
Q
What serves as the adapter molecule specific for each amino acid that binds to specific condons on RNA templates?
A
tRNAs
21
Q
How are amino acids linked to tRNA molecules prior to their incorporation into the protein?
A
enzymatically
22
Q
Which step has the most proofreading properties?
A
replication
23
Q
What organelle has its own set of tRNAs?
A
mitochondria
24
Q
All cells have at least how many tRNAs?
A
32tRNAs
25
What size are tRNA molecules?
about 73-93 base RNA molecules
26
Do tRNAs only recognize one codon?
no some tRNAs can recognize more than one codon (this is called the wobble rule)
27
What is the actual form of tRNA?
folds into a 3D formed L-shaped structure
28
What region is always constant on tRNA?
ACC region
29
What does the D arm of tRNA contain?
dihydrouridine
30
What does the TpseudoC arm of tRNA contain?
the sequence ribothymidine; pseudouridine; cytosine
31
What does the anticodon arm of tRNA contain?
the anticodon
32
What does the amino acid arm of tRNA contain?
contains CCA and binds the amino acid
33
What is typically at the 5' end of most tRNAs?
G
34
How many bases are chemically modified, often by methylation of normal base in tRNA?
8 or more
35
How many base sequences does the anticodon have?
the tRNA anticodon has a 3 base sequence in the anticodon loop on the anticodon arm
36
What does the anticodon of tRNA base pair with?
codon in the mRNA
37
In what orientation does the tRNA and the mRNA base pair?
antiparallel to each other
38
What is the first base of the codon paired with?
the third base of the anticodon and vise versa
39
What is the wobble position?
the first base of the anticodon
40
What is the first step in the wobble hypothesis?
first two bases of a codon always form strong standard base pairs with the corresponding bases of tRNA and confer most of the specificity
41
What is the second step in the wobble hypothesis?
the first base of some anticodons (pairs with third base of codon) determines the number of codons read by a given tRNA
42
What is the third step in the wobble hypothesis?
when an amino acid is specified by several different codons, those codons that differ in either of the first two base require different tRNAs
43
What is the fourth step in the wobble hypothesis?
A minimum of 32 different tRNAs are required to translate all 61 codons
44
What is the first starting codon?
Met
45
Some tRNAs contain the nucleotide inositate which base does it contain and where is it found?
contains the base hypoxanthine at the first anticodon position
46
What nucleotide can hypoxanthine base pair with?
base pair (weakly) with U, C, and A
47
What are ribosomes?
complex protein synthesizing machines
48
Ribosomes are comprised of how many subunits in eukaryotes and prokaryotes?
two subunits a large and a small
49
What is each ribosomal subunit comprised of?
one or more rRNA molecules and many different proteins
50
What is the largest ribosome found in eukaryotes?
23nm in diameter
51
What are the 3 sites that both subunits form on the ribosome?
peptidyl P site
Aminoacyl A site
Exit E site
52
What chromosomes are constantly transcribing new genes?
13,14,21,22
53
Where does protein synthesis occur?
in the cytoplasm
54
What is the S value for the large prokaryote ribosomal subunit?
50S
55
What is the S value for the small prokaryote ribosomal subunit?
30S
56
What is the S value for the ribosome in prokaryotes?
70S
57
What is an S value?
sedimentation value (similar to density)
58
What are the 5 major stages of protein synthesis?
```
activation of amino acids
initiation
elongation
termination and ribosome recycling
folding and posttranslational processing
```
59
How do we activate amino acids?
stage 1: charging the tRNA with its proper amino acid
60
What type of bonds attach amino acids?
ester bonds (carboxylic acid to sugar OH) to the 3' OH of the corresponding tRNA process (called charging the tRNA
61
What is the reaction of charging the tRNA?
an aminoacylation of the 3' OH of the tRNA
62
What is an amino acyl-tRNA synthetases catalyze reaction?
specific for amino acid and one or more corresponding tRNAs
| AA+tRNA+ATP ---> AA-tRNA +AMP+PPi
63
How many classes of enzymes are in all organisms?
2
64
What is charging tRNA?
when an amino acid is put on the tRNA
65
How many high energy bonds are needed to charge the tRNA?
2 AMP and PPi (therefore irreversible)
66
What is the enzyme-bound intermediate in tRNA charging?
aminoacyl adenylate (aminoacyl-AMP) is formed by reaction of ATP and amino acid (carboxyl group anhydride-linked to phosphate; PPi displaced) one high energy bond used
67
What is the second step in tRNA charging?
aminoacyl group transferred to corresponding specific tRNA
68
What are the 4 ways that aminoacyl-tRNA synthetases recognize correct tRNAs?
recognize the anticodon
recognize 10 or more bases in tRNA
recognize one specific base pair
structure features seem to be important
69
What happens once the tRNA is charged by a synthetase?
the aminoacyl tRNA forms a complex with a molecule called EF-Tu that also binds one GTP molecule - now it can bind to the ribosome A site (but first we have to build the initiation complex)
70
What is used in initiation?
a specific methionine tRNA
71
What are the 2 tRNAs for methionine?
one tRNA is used exclusively for initiation and the other for methionines with polypeptide chain
72
What is regular tRNA in bacteria designated to?
tRNA Met
73
What is initiation tRNA in bacteria designated to?
tRNA fMet
74
What is the starting amino acid in bacteria for initiation?
N-formylmethionine
75
What does tRNA fMet do?
it is charged with methionine then a specific enzyme adds formyl group to methionine donated by N10 formltetrahydrofolate
76
How many components are needed for initiation in bacteria?
7
77
What are the 7 components needed for initiation in bacteria?
```
30S ribosomal subunit (contains 16S rRNA)
mRNA
initiation fMet-tRNA fMet
3 initiation factors (IF1-IF3_
GTP
50S ribosomal subunit
Mg2+
```
78
How many steps does it take to assemble and form the initiation complex?
3 steps
79
What is the 1st step in assembling the initiation complex in bacteria?
correctly binding the positions of the initiation codon on the 30S subunit
80
If the codon is bound in a region of 30S what site is the subunit a part of?
subunit is part of the P site
81
How does the initiation codon distinguish where to go?
by the proximity to Shine-Dalgarno sequence
82
Where will the P site of the ribosomal complex be placed?
right over the AUG of our start site
83
What is the 2nd step in assembling the initiation complex in bacteria?
30S subunit, mRNA, IF-1, IF-3 complex is joined by IF-2 (itself bound to GTP) and fMEt-tRNA fMet
84
Where is the anticodon of the initiator tRNA and the initiation codon paired?
at the P site
85
Where does fMet-tRNA fMet bind?
to the P site
86
Where do all other tRNAs bind?
the A site (except for fMet tRNA
87
What is the 3rd step in assembling the initiation complex in bacteria?
large complex binds 50S ribosomal subunit, simultaneously GTP bound by IF-2 is hydrolyzed to GDP, GDP is released by IF-2 and IF-1-IF-3 leave the complex
88
Now the complete ribosome has charged initiator tRNA in complete P site and what is found at the A site?
nothing it is empty
89
How many initiation factors eIFS are involved in protein synthesis of eukaryotes?
at least 9
90
What steps take place in initiation in eukaryotes?
an initiation factor binds to 5' cap and a complex is formed with 40S subunit, mRNA scans for the first AUG codon (which is typically the start of the reading frame) a complete 80S ribosome is assembled using the other eIFs and initiating Met-tRNA Met
91
What is a Kozak sequence?
5'-CCACCAUGG-3' found at many initiation sites
92
How does elongation occur?
stepwise addition of amino acids to polypeptide chain by formation of peptide bonds
93
What is elongation accompanied by?
the movement of ribosome along mRNA from 5' to 3' end reading codons in stepwise manner
94
How many ribosomes are found bound to each mRNA molecule in prokaryotes and eukaryotes?
10-100
95
Can polypeptide chains be synthesized simultaneously?
yes it is a very efficient use of mRNA - polypeptide chains can be synthesized simultaneously using just one molecules worth of genetic information
96
What is required for elongation in bacteria?
```
initiation complex (for first peptide bond)
next aminoacyl-tRNA specified by next codon
elongation factors EF-Tu, Ef-Ts, EF-G
```
97
How many steps are involved in prokaryotic elongation? Are these steps repeated?
3 steps and the process
| yes, repeated for each amino acid residue adding to the growing chain
98
What happens in the 1st step in elongation of prokaryotes?
bind the next aminoacyl-tRNA (as the aminoacyl-tRNA EF-Tu GTP complex) specified by the codon adjacent to the AUG is bound to the A site of the 70 S initiation complex, GTP is hydrolyzed and EF-TuGDP is relased by the ribosome, EF-TuGTP is regenerated and binds to EF-Ts and releases GDP, EF-Tu binds GTP, and releases EF-Ts
99
What is the 2nds step in elongation of prokaryotes?
a peptide bond is formed between the 2 amino acids
100
What is the enzyme catalyzing the second step of elongation in prokaryotes?
peptidyl transferase
101
What type of enzyme is peptidyl transferase?
a ribozyme (instead of a ribosomal protein it is the large subunit 23S rRNA that catalyzed the reaction)
102
What type of energy is provided in the 2nds step of elongation in prokaryotes?
energy is provided by the high energy ester bond between amino acid and tRNA
103
he peptide bond will form between initiation fMet and the second amino acid at which site?
the A site
104
What is base paired to the second codon in the A site of the 30 subnunit it step 2 of elongation in prokaryotes?
a dipeptidyl tRNA
105
Where does the "spent" initiation tRNA base pair to the aUG on the 30S subunit?
in the P site
106
Step 2 of elongation a peptide bond changes formation what does this result in?
a change in the interaction of tRNAs with the 50S but not the 30S subunit
107
Where does the deacylated initiation tRNA move to?
moves into the E (exit) site on the 50S subunit, stays in P on 30S subunit
108
Where does the new dipeptidyl tRNA move to?
moves to the P site on the 50S subunit but stays in the A site on the 30S subunit
109
What happens to the position of the anticodon in the 30S ribosome site?
it is unchanged
110
What is the 3rd step in elongation in prokaryotes?
translocation: ribosome moves along mRNA precisely one codon towards the 3' end, movement is mediated by elongation factor G (EF-G) and GTP
111
What is the EF-G GTP bound by?
ribosome
112
What happens when GTP is hydrolyzed?
EF-G and GDP are released (energy moves the ribosome)
113
How many tRNAs can be bound at any given time?
2
114
What does the movement of the ribosome cause to occur?
shifts the dipeptidyl tRNA from the A site on the 30S subunit to the P site (now all in the P site)
115
What does the spent tRNA cause to occur?
shifts from the P site on the 30S to the E site (all in E) and the A site is empty and ready for next aa-tRNA
116
When the next aa-tRNA begins the next round of elongation what happens to the A and E sites?
filling of the A site induces release of spent tRNA from the E site
117
Can the A and E sites be occupied simultaneously?
no
118
How many steps and how many elongation factors are required for elongation in eukaryotes?
3 elongation factors and 3 steps
119
Why use two steps to move tRNAs in elongation?
anchors one end of tRNA while the other moves during peptide bond formation and translocation, also prevents premature release of peptidyl-tRNA (tremendous waste of energy) and enhances processivity
120
What is the proofreading on the ribosome limited to?
codon-anticodon interactions (identity of amino acid not checked)
121
When does the proofreading occur?
while EF-Tu GTP and EF-Tu GDP bound (milliseconds) codon anticodon base pairing is checked
122
What happens if the incorrect tRNA codon pairs?
they would dissociate during this time
123
What is a way that speed and fidelity are optimized?
slower speed = higher fidelity and vice versa
124
What happens during termination?
elongation continues until ribosome encounters in frame stop codon in A site
125
One of three termination factors bind to termination codon what are they?
RF1 for UAG, UAA
RF2 for UGA, UAA
RF3 - not known
126
In termination why is peptidyl transferase induced?
to transfer peptide chain to water resulting in the release of peptide chain from the ribosome (uncharged tRNA in P site reelased and 30S and 50S subunit dissociate)
127
How many termination factors do eukaryotes have?
just one
128
What 2 things are tightly coupled in bacteria?
transcription and translation
129
Why is protein synthesis energy expensive?
charging tRNA takes 2 high energy bonds
first elongation step GTP--> GDP
translocaitons tep: GTP--> GDP
therefore at least 4 high energy bonds are expended per peptide bod=122kj/mol foron e 21kj/mol peptide bond
130
Energy is used for specificity. What is specificity?
used to maintain proper alignment of a codon, tRNA and growing end of the polypeptide chain
131
What is the overall error rate for protein synthesis?
1:10,000 amino acids (Much higher than DNA replication)
132
How correct are the chains typically?
about 90% correct
133
How correct are the chains that smaller proteins?
about 99%
134
Why is the accuracy not higher?
because it would greatly slow protein synthesis down and would only gain modest accuracy
135
What 2 things can inhibit translation in prokaryotes and eukaryotes?
antibiotics and toxins
136
Does a prokaryotic inhibitor harm eukaryotes?
there are sufficient differences between the organisms so a prokaryotic inhibitor is relatively harmless to eukaryotes
137
What are the 7 inhibitors of translation?
```
tetracyclines
chloramphenicol
streptomycin
aminoglycosides
cycloheximide
diphtheria toxin
ricin
```
138
What does tetracycline do?
binds to prokaryotes A site; blocks aa-tRNA binding and is useful in treating periodontal disease (stains teeth)
139
What does chloramphenicol do?
blocks peptidyl transfer in prokaryotes and mitochondria, not eukaryote 80S ribosomes
useful to treat rickettsial infections (rocky mountain spotted fever, typhus) very toxic ex: blood disorders such as aplastic anemia
140
What does streptomycin do?
binds S12 of 30S ribosome
low concentrations cause misreading of code
high concentrations inhibit initiation
141
What does aminoglycosides do?
block 50S ribosome binding to initiation complex
142
What does cycloheximide do?
blocks eukaryotes 80S peptidyl transferase not prokaryotes or mitochondria
143
What does diphtheria toxin do?
catalyzes ADP ribosylation of eEF2 inactivating it
144
What does ricin do?
extremely toxic protein from castor bean - inactivates eukaryotes 60S subunit by depurinating A residue of 28S rrNA
