tRNA and Translation
Shine Dalgarno sequence is located at
-10 region and on the mRNA there is purine rich region of AGGAG, which is complementary to the pyrimidine rich sequence on the 16S rRNA UCCUC
Translation initiation uses
1 GTP
the ribosome is assembled around the mRNA with the loss of
1 GTP; IF2
What are the two types of Aminoacyl-tRNA synthetases and their differences?
1. Class I synthetases have to Rossmann fold used to bind ATP; Class II have different ones 2. Class I synthetases must recognize their anti-codon while Class II synthesis do not need that 3. Class I synthetases aminoacylate the 2'OH group while the Class II aminoacylate the 3'OH group. In the end, transesterificaiton leads to 3'OH amino acylation 4. Class I synthetases tends to work for larger and hydrophobic amino acids
What are the loops of tRNA structure?
1. D loop: dihydrouridine 2. T loop: pseudouridine 3. Anticodon loop 4. Variant loop
Decoding steps
1. EFTu, GTP and aminoacyl tRNA forms a complex and is docks at the A site. 2. The complex is loaded onto the A site with the hydrolysis of GTP to GDP and the release of EFTu-GDP complex
In translation: 3 factors were used altogether,
1. EFTu- recognition 2. EF-Ts- exchange factor 3. EFG-release of uncharged tRNA from P-site
How is EFtu-GDP complex recycled?
1. GDP is replaced by EFTS 2. EFTS is replaced by GTP. so EFTu-GTP forms again for next decoding
Translation initiation in prokaryotes
1. IF3 binds to 30S leading to the dissociation of 30S/50S complex 2. IF2, GTP, fmettRNA, mRNA forms a ternary complex, with IF-1 joining and binding of 30S with IF3 to the shine Dalgarno sequence forms the 30S initiation complex 3. IF 3 and IF 1 leaves, this allows for the 50S to bind. the binding of 50S triggers GTP hydrolysis catalyzed by IF2, leading to 30S to change its conformation. So, IF2, GDP and Pi leaves, formations of 70S initiation complex
translation
1. Post-translocational state: E and P sites are occupied. The binding of EFTu-GTP leads to the release of the E site tRNA. At the same time, the A site tRNA enters by base paring. The acceptor stem stay out of the 50S site because of the EF-Tu binding 2. GTP hydrolysis of EFTU binding leads to the release of EFTU and the entry of the acceptor stem of tRNA to the 50S A site. 3. Transpeptidation reaction: there peptide is transferred from the P site to the A site catalyzed by 50S by desolventing and proximity (A2486 and 2'OH of ribose) 4. pre-translocational state: the peptide translocates from the A site to the P site on 50S because they have affinity for the E site on 50S while the codon-anticodon interactions on 30S remains the same 5. EFG binding and hydrolysis of GTP drive the translocation-- movement of both tRNA and mRNA by exactly 3 nucleotides 6. Translocation process is spontaneous, however with the help from EFG and GTP hydrolysis, the process is much faster
What are the 3 modifications of the primary transcript of tRNA?
1. RNase P removes 5' extra nucleotides 2. addition of universal CCA nucleotides at the 3' end 3. modification of other nucleotides, especially ones close to the anticodon loop. 2 types of modification A, methylation B. pseudouridine
What are the arms of the tRNA structure?
1. acceptor arm 2. Dihydrouridine arm 3. t arm 4. anticodon arm 5. variant arm
What is the 3 key processes in translation elongation?
1. decoding: selecting the correct aminoacyl-tRNA to the mRNA codon 2. transpeptidation: peptide bond formation-- no energy needed! 3. translocation: peptidyl tRNA moved from A site to P site
Simple steps for translation initiation
1. dissociation of 30S/50S 2. formation of 30S initiation complex 3. formation of 70S initiation complex
What are the two steps of amino acid recogniztion?
1. first and inaccurate synthetic step (charging) 2. eliminates misactivated non-cognate amino acids
What are the two steps of proofreading of tRNA syntheses?
1. hydrolyze the adenylate (AMP) before it goes on to tRNA (activated amino acid) 2. hydrolyze the tRNA-amino acid ( the charged tRNA)
Decoding proofreading is a two step process
1. recognition at codon/anticodon position 2. aminoacyl tRNA fully moves into the A site after the hydrolysis of GTP by EFTU This means GTP hydrolysis is going to be much faster when it is in the correct tRNA mRNA pair than if it is the incorrect pair; or GTP hydrolysis is faster for cognate tRNA== Kinetic proofreading
What is the function of ribosome?
1. recognize mRNA and tRNA (30S) 2. catalyze peptide bond formation (50S) 3. ribosome undergoes movement so that it can translate sequential codons
How did they figure out the actual code?
1. synthesis of oligonucleotide: pulled down radio labled AA, modified tRNA by binding to nitrocellulose bound ribosome and then figured out which mRNA corresponds to Amino acid 2. cell free translation system: used just mRNA, ribosomal components and tRNA prove that all you need to code was mRNA and don't need the original DNA
Aminoacyl-tRNA synthetases
1. the amino acid reacts with ATP to form aminoacyl-AMP or aminoacyl-adenylate The subsequent hydrolysis of pip makes this reaction irreversible 2. The amino acid, which has been activated by its adenylation, reacts with tRNA and forms aminoacyl-tRNA ( charged)
Translation Termination
1. when the ribosome moves to the stop codon, the release factor RF1 or RF2 binds to the A site and triggers the hydrolysis of the peptide (H2O is nucleophile) from the tRNA at the P site 2. The binding of GTP-RF3 and the subsequent hydrolysis leads to the release of RF1/2 from the active site 3. RF3-GDP leaves 4. The binding of EFG-GTP and Ribosomal Release factor: RRF 5. Hydrolysis of GTP by EFG drives RRF to the P site and the release of tRNA from P and E sites 6. Finally, the RRF, mRNA, EFG are released from the 70S, which is inactive again and needs to be reinitiated again
primary tRNA transcript is how many nucleotide
108
how many invariant positions does tRNA have?
15
30 S is made of
16S and 21 proteins
In X ray structure, the Shine Dalgarno sequence--located close to the E site bases pairs with the
16S rRNA
Every amino acid after that uses
2 GTP because it does not need to assemble to initiation complex again
Termination uses
2 GTPs
Decoding has how many protein components
2! EFTu and EFTS
50S is made of
23S and 5S and 36 proteins
How many reading frames are there?
3
A1492, A 1493, and G530 are
3 invariant positions on the 30S subunit ribosome; universally conserved ribosomal bases--> always there
Prokaryotic ribosome have two subunit :
30S and 50S
For the 50S
33% is protein by mass and 66% is rRNA
For the small 30S,
40% is protein and 60% is RNA
mRNA is read from
5' to 3'
How long is the primary sequence of tRNA normally around?
54 to 100 nucleotide, with most of them around 76 nucleotides
How long are the legs?
60A
Prokaryote ribosome-- whole ribosome is
70S
How far apart are the legs?
76A
mature tRNA transcript is how many nucleotide
78
how many semi-invariant positions does tRNA have?
8
U binds to
A and G
The ribosome have 3 sites
A-site: amino acyl tRNA binding site P site: peptidyl-tRNA binding site E site- exit site, deacylated tRNA
There are 2 start codons
AUG and GUG
Protein synthesis starts with
AUG codon, which encodes Met
You add on the
C term first of the amino acid
I binds to
CAU
How do Class I and II tRNA synthetases approach their tRNA?
Class I and II tRNA synthetases approach their tRNA with the inside of their L-shape; they are not recognizing the anti-codon or amino acid specifically; they are binding the whole tRNA; they recognize a big interface
Translocation needs EFG. How many domains does EFG have?
EFG have 5 domains. Two of them are very similar to EFTu-GTP binding domain. The other three domains mimic the tRNA bound to EFTu
You always want to load into the A site, so the aminoacyl tRNA must be in a complex with
EFtu and GTP for recognizing mRNA
What is the error rate of aminoacylation fidelity?
Error rate: 1:10000 turnovers
N formylmethione-tRNA (met)
Formyl group added to the amino group of the methionine
C binds to
G
Translation uses what type of energy?
Gprotein energy! GTP
What are the initiation factors for translation?
IF-1, IF-2, and IF-3
GTP hydrolysis is catalyzed by what in translation initiation?
IF2
What does the 3-D structure of tRNA look like.?
L shape
Proteins are produced from the
N terminal to C terminal
What are the key interactions around the active site of the ribosome?
RNA-RNA interactions
How does 50S catalyze peptide bond formation? peptidyl transferase reacion
Rate enhancement by desolventing and proximity and orientation The most important interactions A2486 with its N3 hydrogen bonding with A site amino acid group and the P site 2'OH group on the ribose when delta H and delta S are measured, it was found that delta S is the main contribution= "entropy trap" Thus, Ribosome enhance the rate (10^7) by properly positioning and orienting its substrate and exclude water from the active site
What removed the extra 5' nucleotide on tRNA?
Rnase P
Ribosome helping the correct recognition between codon and anticodon
The orientation of A1492, A1493, and G530: when the correct basepair is formed, the Adenines are flipped in and the ribosome kind of clamps down on it and helps with the recognition. It clamps down and help form the competent complex
Transpeptidation
The transfer of peptide from P site tRNA to A aminoacyl-tRNA does not need any energy input. This process is entirely catalyzed by 50S subunit of the ribosome.
A binds to
U
G binds to
U and C
There are 3 stop codons
UAA, UAG, UGA
Proteins in general has
a global structure with positively charged tails.
EFTU is highly
abundant and almost all tRNA is EF-Tu associated
What forms the legs of the 3-D structure of tRNA?
acceptor arm and t-arm form one leg anticodon arm and D arm forms one leg
Codons that differ in 1st and 2nd positions, will
acquire different tRNAs
suppression is when you
add another mutation to go back to the correct frameshift
tRNa is an adaptor molecule between an
amino acid and nucleic acid; translate between coded DNA and amino acid
acceptor stem is going to get the
amino acid on it
With the correct base paring, conformation changes associated with GTP hydrolysis by EFTu will swing the acceptor arm of the tRNA to 503. However, with the wrong base paring
aminoacyl-tRNA simply dissociates from the ribosome
formyl group is
an aldehyde group
What percent of tRNA was modified?
around 25% nucleotide are modified
How many tRNAs do you usually have for codons?
around 32 tRNAs ( 31 to code and 1 to initiate) to cover all 61 codons
What is the D loop named after?
because it contains dihydrouridine
What triggers IF2 to hydrolyze GTP
binding of 50S subunit
Class I takes
bulky hydrophobic amino acid
If these bases are in the first or wobble position of anticodon C A G U I
c binds to G A binds to U G binds to U and C U binds to A and G I binds to C A U
What is the function of 50S
catalyzes peptide bond formation
The positively charged tails of proteins are
conserved and used to interact with rRNAs
Another GTP is used to
decode; EFtu
Example of Cystic Fibrosis sickness
deletion of T keeps you out of reading frame
Cells also have silent mutations that
do not change AA incorporated
23 S rRNA does not have
domains
Overall the aminoacyl-tRNA synthetase is a
energy consumption process and the reaction is driven by PPi hydrolysis. This is a transesterification reaction
Ribosome has many proteins but the active site is
exclusively made of RNA. Proteins are located on the surface
A1493 stabilized the
first base paring by interacting with them through the minor groove
if the second letter in the codon is a purine, it is
for polar amino acids
For the second letter in the codon, if it is a pyrimidine (U or C), it is
hydrophobic
Kinetic proofreading
if I put the wrong tRNA in, GTP hydrolysis is slow and lets the tRNA to have a change to move out and the correct one to move in. This is a lag that allows the correct tRNA to pair. this is proofreading by slowing down the incorrect process and speeding up the correct process
Where is the wobble position in the mRNA and tRNA?
in the mRNA, its the 3rd position of the codon In the tRNA, it is the 1st position of the anticodon
frameshift
insertion or deletion of nucleotides
A2486 is
invariant is working together with the 2'OH group of the ribose, which is part of the tRNA on the growing chain. Those two are holding the amino group right in line to attack that carbonyl
What does IF1 do?
it occupies the A site to prevent the loading of N-formyl-methione-tRNA to the A site
What does IF3 do?
it prevents binding of tRNA other than N-formyl-met-tRNA
In order to make a protein, how much energy is needed?
it takes a lot of ATP to make a protein-- in fact-- 30% to 50% of energy consumption in a growing cell forms peptide bonds at rate greater or equal to 10S-1
For Class I synthetase, it initially acylate the 2'OH first but
it will eventually migrate to the 3'OH position because thats the most thermodynamic stable
AMP is a good
leaving group
bacteria have
less than 40 tRNAs to bind all 61 codon
What is the function of 30S?
mRNA binding and codon-anticodon interactions
Class II is going to use the
major groove of the acceptor stem
Class I uses the
minor groove of the acceptor stem
Are most of tRNA solvent accessible?
most of tRNA molecules are solvent inaccessible, except for the acceptor arm and anti codon arm
Where does a lot of modification of tRNA occur?
near the anticodon loop
the third position is more flexible and can have many
non standard H-Bonds
Genetic code is
nonoverlapping, degenerate, and triplet code
genetic code is degenerate and
nonrandom
If you insert 3 nucleotide, do you still cause frameshift?
nope
Is the genetic code universal?
nope, the genetic code is not universal. some codons used in mitochodria or ciliated protozoa are different from standard genetic code
Is the AUG or GUG start codon enough to start translation initiation?
nope. you need shine dalgarno sequence
Sickle Cell anemia has a
point mutation
Ribosome is the site for
protein synthesis; it is RNA-protein complex
T arm is named because it contains
pseudouridine
Both A1492 and G530 stabilized the
second base paring by interacting with them through the minor groove
16S has
several domains: 5'-domain (body), the central domain (the platform) and the 3' domain
change in the first letter encodes a
similar amino acid; extra protection step
Class II takes
small polar amino acid
what does degenerate mean
some amino acid are specified by more than one codon
reading frame
succeeding nucleotides are read as codons
What is pseudouridine (Psi)?
swap the attachment site of the ribose sugar from the nitrogen to carbon; Forms c-c bond
the first tRNA that comes out is called
tRNA(tyr) primary transcript
the amino acid is always going to be attached to what part of tRNA?
the 3' oH of tRNA at the Adenine because of the universal CCA sequence
What holds the 3 D tRNA structure together?
the 3-D structure are held by interactions from the invariant (15) and semi-invariant (8) nucleotides; many of them are in nonstandard H-bonds
What is the catalytic unit of Rnase P?
the RNA is the catalytic unit
polysome
the association of multiple ribosomes with mRNA leads to the formation of the structure called poly ribosome or polysome
Peptide bond formation
the peptide is transferred from the p site tRNA to the amino acid on the A site tRNA. Your chain is now in the A site. So, then the peptide tRNA is then translocated from the A site to the P site
What does the protein unit do on RNase P?
the protein unit is to stabilize complex by neutralizing the charge-charge repulsion
What does the secondary structure of tRNA look like?
the secondary structure of tRNA is like a cloverleaf structure
What does the start codon tell us?
the start codon tells us where to start the reading frame
How can you explain why Class I acylate the 2'OH first?
the structural difference explains why class 1 acylate 2'OH first. The class I 3' end forms a hair pin turn when it enters the syntheses active site; Class II continues its helical turn
Translation energy:
there is 10 "ATP equivalents" per amino acid residue 30-50% goes to protein synthesis
Why is rate enhancement form not acid/ base catalysis?
this is because the pKa of N3 of A2486 is 3.5 and is not good enough to deprotonant the amino group (pKa=10) (needs to be within 2-3 pH units, like around pH 7) Rate enhancement comes around proximity and desolventing
The codon usage is not equally distributed among all available of 61 codons
this is correlated to corresponding tRNA concentrations
suppressor
two mutations that cancel the effects from each other
Use of another GTP to release
uncharged tRNA from P site; EFG
the first two positions in the tRNA anticodon codon interactions have normal
watson crick H bonding
The third base paring is reinforced though the minor groove interaction by G530 but this interaction is
weak and allows for wobble position
How does A 1492, A 1493, and G530 of the 30S subunit help?
when the EFtu comes in , you are going to have the correct competent recognition and thats going to help to get the correct conformation for the hydrolysis of EFTu
The third position is the
wobble position. It can vary but still give the same amino acid
is the translocation process spontaneous?
yes, it will happen no matter what but with the help from EFG and GTP hydrolysis, the process is much faster
DO you have enough tRNA for all of those codons?
you don't always have the same number of tRNAs available for each of the different codons. Some codons are used more rarely than others, which leads to slight pauses in translation( good for proofreading)