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)