The Translation Process Module 4 Lecture 1 part 2

Why do you not need a GEF with EF-G?

Because EF-G has a higher affinity for GTP not GDP so GDP just falls off and rebinds GTP. Can reload itself basically.

How quickly can the ribosome incorporate amino acids?

at about 12 or 15 AA per second

What does Ef-tu do?

an élongation factor that brings in each Amino acyl tRNA

What happens after the 50S subunit joins during initiation of translation in prokaryotes?

After the 50S subunit joins, it turns out the the fMet tRNA was bound in what would become the P site once you form the 70S initiation complex. Every other incoming tRNA will always bind to the A site.

What happens energy wise during termination of translation?

-- 1 phosphate bond per polypeptide chain (GTP---GDP by RF-3)

What happens energy wise during recycling of translation?

-- 1 phosphate bond per polypeptide chain (GTP--GDP by EF-G)

What happens (energy wise) during Initiation of translation?

-- 1 phosphate bond per polypeptide chain (GTP--GDP by IF-2)

What happens energy wise during charging in translation

-- 2 phosphate bonds per amino acid in protein (ATP---AMP by AA-tRNA synthetase)

What happens during elongation energy wise?

-- 2 phosphate bonds per peptide bond formed in protein (GTP-- GDP by EF-Tu and 1 by EF-G) --------note for n amino acids, there are n-1 peptide bonds

What are the specific steps of initiation of translation in PROKARYOTES?

1. Binding of IF-3 to the 30S subunit when the 50S and 30S subunits are apart and this prevents the 50S subunit from binding and getting in the way during initiation. 2. mRNA that has to bind to the ternary complex (3 components to it- IF-2 with GTP and the fMet tRNA are in this ternary complex). So IF-2 delivers the fMet tRNA and the GTP so it can bind to the mRNA. 3. IF-1 assists IF-3 binding and blocks A site during assembly of initiation complex.

What are the very basic steps involved in elongation of translation?

1. Decoding 2. Transpeptidation - formation of peptide bond 3. Translocation- moving the peptidyl tRNA over to the P site from the A site.

What 2 things are GTPases in the elongation part of translation?

1. EF-G 2. EF-Tu

What are the elongation factors for translation in prokaryotes?

1. EF-Tu- Binds aminoacyl-tRNA 2. EF-Ts- Displaces GDP from EF-tu 3. EF-G- Promotes translocation thru GTP binding and hydrolysis

What are the initiation factors for prokaryotes in translation?

1. IF-3- binding to the 30S subunit prevents 50S subunit from joining 2. IF-2- delivers fMet-tRNA and GTP to 30S subunit in ternary complex 3. IF-1- assists IF-3 binding and blocks A site during assembly of initiation complex ****** THESE ARE IMPORTANT FOR THE FORMATION OF THE 30S INITIATION COMPLEX*****

Go through the step by step process during translation in prokaryotes when the 30S initiation complex transitions to the 70S initiation complex. (2nd big step of initiation of translation)

1. If everything looks good and there are codon- anticodon interactions between the fMet tRNA and the AUG start codon GTP will get hydrolyzed into IF-2 and GDP. 2. After GTP gets hydrolyzed into IF-2 and GDP you will then release IF-1 and IF-3 and you are ready for the 50S subunit to come in and join 3. After the 50S subunit joins, it turns out the the fMet tRNA was bound in what would become the P site once you form the 70S initiation complex. ******Every other incoming tRNA will always bind to the A site. *****

What are the phases of translation?

1. Initiation 2. Elongation 3. Termination 4. Recycling

What are the release factors in prokaryotes for termination and recycling of translation?

1. RF-1: Recognizes UAA, UAG codons 2. RF-2: recognizes UAA, UGA codons 3. RF-3: GTPase that facilitates RF-1 and RE-2 release after polypeptide release

What are the 2 proofreading steps during elongation specifically?

1. The first is if its totally off, like you don't have any correct base pairing between the codon and anti codon and it just falls right out. It does NOT involve hydrolysis of GTP. 2. The 2nd proofreading step is if 2 out of 3 or all 3 are base pairing properly and the cell needs to check and make sure its correct, (FINE TUNED), hydrolysis of GTP by EF-tu and that has the ribosome stick some bases of the rRNA right into that groove between the codon and the anti codon to double check it. (LIKE LOCK And KEY). If it is correct then EF-tu will dissociate and you are ready to make the peptide bond. If its wrong, the amino acyl tRNA and EF-tu will fall off and you try again.

What are the specific steps involved in elongation of translation?

1. You already have growing polypeptide chain on the peptidyl tRNA in the P site, then you have the A site where the newly bound amino acyl tRNA has come in. 2. To form the peptide bond you transfer polypeptide chain from the P site over to the A site which added a 4th amino acid (you have 3 before) 3. Translocation of the ribosome (2 step process ) this is so you can move the next codon into the A site. This involves shifting the top part or large subunit then you shift the small subunit. You move both over 3 bases (one codon but one site) and the next codon is in the A site. Then the process is restarted until you hit a stop codon.

GO thru the step by step process of translation termination in prokaryotes.

1. You get a stop codon (lets say UAG codon) in the A site. 2. RF-1 binds to the A site at the stop codon and if recognition occurs, you get hydrolysis of this last ester bond and release of the polypeptide chain 3. RF-3 (hydrolyzes GTP and this results in popping off of RF-1 and RF-2) comes in and pops off RF-1 in this case (could be RF-2 also) 4. RRF and EFG breaks everything down so that you are ready to go back and initiate again on a new mRNA- demolition crew essentially

Describe the incorporation of an AA into a growing polypeptide chain on a ribosome step by step. (include mechanism of bond formation)

1. the peptidyl tRNA (at the P site) attached to C-terminus of the growing polypeptide chain 2. The aminoacyl-tRNA with a single amino acid on it binds to the A site (amino acyl) 3. The amino group on the aminoacyl tRNA carries out a nucleophilic attack on the ester bond of the peptidyl chain. The tRNA molecule is freed from its peptidyl linkage 4. After the nucleophilic attack, the polypeptide chain that was on the peptidyl tRNA gets transferred over to the amino acid on the aminoacyl tRNA via a covalent bond. This results in the new peptidyl tRNA molecule attached to the C terminus of the growing polypeptide chain. The polypeptide chain essentially shifts from the p site tRNA to the A site tRNA and you are adding an amino acid to it.

What is the subsequent disassembly of post termination complex mediated by?

2 factors: 1. Ribosome Recycling Factor (RRF) 2. EF-G

How many codons encode an amino acid?

61 codons; due to 3 top codons. They can range from 1 to methionine to 6 for leucine for example.

What is EF-ts

A GTP exchange factor or a GEF; When you hydrolyze GTP with EF-Tu you have EF-Tu- GDP, the affinity of EF-Tu is higher for GDP than GTP, so you need a protein to help pop off the GDP to add GTP (so you can start the process over again - recharge) and this protein is called EF-Ts

What do you call the process of linking an amino acid to a tRNA A. Charging B. Amino acylation C. Fission D. Fusion

A. Charging or B. Amino acylation

What is the 2nd step in the transition of the 30S initiation complex to the 70S initiation complex during initiation of translation in prokaryotes?

After GTP gets hydrolyzed into IF-2 and GDP you will then release IF-1 and IF-3 and you are ready for the 50S subunit to come in and join

Which is not a stage of translation: A. Initiation B. Replication C. Elongation D. Termination E. Recycling

Answer: B Replication

Name the tRNA binding sites on the ribosome: A. ABO B. RPG C. APE D. All of the above

Answer: C, APE (amino acyl, Peptidyl and exit sites)

What are key differences between tRNAs and mRNAs? A. tRNAs have more unusual base pairs than mRNAs B. tRNAs have a more distinct uniform structure than mRNAs C. tRNAs are generally shorter than mRNAs D. All of the above

Answer: D all of the above

How many codons are present in the genetic code? A. 4 B. 16 C. 32 D. 64

Answer: D; 64 codons in the genetic code

Where is the PTC located in regards to the tRNAs?

Between the A site and P site tRNAs. This is the site that makes the chemical bond between the nascent chain and the next amino acid.

How do R-proteins facilitate ribosome function?

By transmitting info between different rRNA structures. These proteins are actually connecting different RNA domains so that you can couple conformational changes in one domain to another domain and this helps facilitate the translation process and is a kinetic stimulator. (The pic shows this with the yellow tail snaking throughout the different domains within the RNA)

How are proteins critical in the ribosome?

Catalytically, without them ribosomes would form like 1 peptide bond a day, not efficient

What factors are NOT involved in translation? A. IF1, IF2, And If3 B. EfTu, EfTs, EF-G C. RF-1, RF-2, and RF-3 D. Pol I, PolII, Pol III

D, Pol I, Pol II, Pol III

Which GTPase in the elongation part of translation has a higher affinity for GTP?

EF-G

What facilitates the translocation of the ribosome?

EF-G GTP binds to A site

Which GTPase in the elongation part of translation has a higher affinity for GDP?

EF-Tu

Blank is a G protein and GTP hydrolysis with G proteins almost always involves what?

EF-tu is a G protein and GTP hydrolysis with G proteins almost always involved in a protein conformational change. You hydrolyze GTP you disrupt that tRNA binding site and that tRNA falls off.

When do you move the tRNA over to the exit site?

Each time you make a new peptide bond addition

What do you hydrolyze GTP with in the elongation process during translation?

Ef-Tu

T or F. IF-3 is a GTPase

FALSE, IF-2 is a GTPase

T or F. R proteins contribute directly to peptide bond formation

FALSE, There are no protein side chain atoms wihtin 18 angstroms of the PTC, so r-proteins DO NOT contribute to peptide bond formation. They are important in catalysis.

T or F. Every incoming tRNA first binds to the P site.

FALSE, only the first fMet tRNA binds to the P site, every other incoming tRNA will always bind to the A site (these will be amino acyl tRNAs)

T or F. The ribosome translocates 2 nucleotides at a time

FALSE, this would result in a frameshift mutation. Must be 3

When you hydrolyze GTP with EF-Tu you have EF-Tu- GDP, the affinity of EF-Tu is higher for what? What does this mean you need?

GDP than GTP, so you need a protein to help pop off the GDP to add GTP (so you can start the process over again - recharge) and this protein is called EF-Ts

What does IF-1 do?

IF-1- assists IF-3 binding and blocks A site during assembly of initiation complex. This is important for the formation of the 30S initiation complex in prokaryotes

What does IF-2 do?

IF-2- delivers fMet-tRNA and GTP to 30S subunit in ternary complex. This is important for the formation of the 30S initiation complex in prokaryotes

What does IF-3 do?

IF-3- binding to the 30S subunit prevents 50S subunit from joining prematurely. This is important for the formation of the 30S initiation complex in prokaryotes

What has to be true in order to GTP to get hydrolyzed during initiation of translation in prokaryotes?

If everything looks good and there are codon- anticodon interactions between the fMet tRNA and the AUG start codon GTP will get hydrolyzed into IF-2 and GDP.

What is the first step in the transition of the 30S initiation complex to the 70S initiation complex during initiation of translation in prokaryotes?

If everything looks good and there are codon- anticodon interactions between the fMet tRNA and the AUG start codon GTP will get hydrolyzed into IF-2 and GDP.

What happens after the initial proofreading step during elongation of translation?

If everything with the amino acyl tRNA base pairing looks good then we would hydyolyze GTP to form GDP and then an additional proofreading step would occur. Again incorrectly base paired tRNAs preferentially dissociate.

What does the 2nd proofreading step in elongation of translation involve?

In the A site, A1492 and A1493 are in helix 44 of the 16s rRNA and are 2 strands of helices. once the tRNA binds to start the codon-anticodon interactions with the mRNA strand, these 2 bases (A1492 and A1493) flip out right into this groove to where they are touching the pairings making sure there is no bulge (mispairing) or anything. This accompanies GTP hydrolysis, If everything checks out, then EF-Tu will leave and amino acyl tRNA will be be accepted and you will have this amino acid. If one of these is bulging and it decides its not good, this amino acyl tRNA will get kicked out, you have already hydrolyzed GTP but Ef-Tu GTP will dissociate anyways and you try all over again. This is fine tuning of proof reading.

When does IF-3 bind? Where does it bind?

It binds to the 30S subunit when the 50S and 30S subunits are apart and this prevents the 50S subunit from binding and getting in the way during initiation of translation in PROKARYOTES.

What is the business end of the ribosome and where is it located?

It is the Peptidyl Transferase Center (PTC) it is found in the Large (50S) Subunit.

What is the order of the functional units in the small subunit from left to right? What does this correspond to?

Order is A, P and E in small subunit and this corresponds with the mRNA channel (where mRNA binds) A and P site tRNA will be base paired to the mRNA codons. The E site is not paired because he is getting ready to exit.

What makes the ribosome a ribozyme?

There are no protein side chain atoms within 18 angstroms of the PTC, so r-proteins DO NOT contribute to peptide bond formation. They are important in catalysis.

What kind of movement happens between the interface of the 2 ribosomal subunits that is thought to help move mRNA and tRNAs thru the ribosome during protein synthesis?

Ratchet like

What does RF-3 do?

Release factor that is a GTPase that facilitates RF-1 and RE-2 release after polypeptide release

What does RF-1 do?

Release factor that recognizes UAA, UAG codons

What does RF-2 do?

Release factor that recognizes UAA, UGA codons

T or F. During every step of translation either ATP or GTP is being consumed

TRUE

T or F. Recycling is a critical step of translation

TRUE

T or F. The ribosome is a ribozyme

TRUE

T or F. The E site tRNA is not base paired with the mRNA.

TRUE, because its getting ready to exit

T or F. The 50S and the 30S subunit are coming together as often as they are coming apart.

TRUE; they are in equilibrium.

What are the residues important for PTC's mediation of peptide bond formation? Where specifically are they located in regards to functional sites?

The PTC mediates peptide bond formation through the action of residues in 23S rRNA. This reaction occurs between the A and P site but 23S rRNAs can be found surrounding these sites and throughout the large 50S subunit of the ribosome. (Orange and white on pic)

What is the point of the double proofreading step during elongation of translation?

To make sure that you have the correct codon anticodon interaction so that your polypeptide has the right amino acid sequence in it.

Blank is a repeating cycle as additional mRNA codons are translated

Translation Elongation

What is the minimum number of high energy phosphate bonds that would be consumed by a protein of 100 amino acid during translation?

`395

What happens if an incorrect based paired tRNA comes into the A site during elongation of translation in prokaryotes?

a proofreading step in which incorrectly base paired tRNAs preferentially dissociate.

What does EF-G do?

facilitates translocation to next codon during elongation. Elongation factor.

How does the amino acyl tRNA come in during elongation of translation in prokaryotes?

in a ternary complex with EF-Tu and GTP and binds in the A site. Basically the cell is sampling different amino acyl tRNAs bound to EF-tu and GTP until it finds one that is the correct anti codon codon interaction.

Where does the reaction for making the protein occur?

in the interface between the large and the small ribosomal subunits

What is the ternary complex in prokaryotes during initiation of translation?

it is a complex with 3 components to it: IF-2 with GTP and fMet tRNA. IF-2 delivers the other 2 components to the 30S subunit so it can bind with the mRNA during initiation of translation in prokaryotes.

What facilitates the termination and recycling processes in translation?

release factors and recycling factors

What molecule serves as an adaptor during translation?

tRNA

What does the PTC do and how does it do this?

the PTC mediates peptide bond formation through the action of residues in 23S rRNA

What is the site that makes the bond between the nascent chain and the next amino acid?

the PTC; Peptidyl Transferase Center

What is the transpeptidation reaction?

transfer of nascent chain from P site to A site. You made the peptide bond here.