Prok Translation Final

Amino-acyl tRNA synthetases: One synthetase for each amino acid a single synthetase may recognize multiple tRNAs for the same amino acid Two classes of synthetase. Different 3-dimensional structures Differ in which side of the tRNA they recognize and how they bind ATP Class I - monomeric, acylates the 2’OH on the terminal ribose Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val Class II - dimeric, acylate the 3’OH on the terminal ribose Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

tRNAs are activated by amino-acyl tRNA synthetases

Two levels of control to ensure that the proper amino acid is incorporated into protein: 1) Charging of the proper tRNA

2) Matching the cognate tRNA to the messenger RNA

Incorporation of amino acids into polypeptide chains I

Incorporation of amino acids into polypeptide chains II

In addition to the APE sites there is an mRNA binding groove that holds onto the message being translated

Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, & IF-3.  IF-3 binds to the 30S ribosomal subunit, freeing it from its complex with the 50S subunit.  IF-1 assists binding of IF-3 to the 30S ribosomal subunit. IF-1 also occludes the A site of the small ribosomal subunit, helping insure that the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.  IF-2 is a small GTP-binding protein. IF-2-GTP binds the initiator fMet-tRNAfMet & helps it to dock with the small ribosome subunit.

 As mRNA binds, IF-3 helps to correctly position the complex such that the tRNAfMet interacts via base pairing with the mRNA initiation codon (AUG). A region of mRNA upstream of the initiation codon, the Shine-Dalgarno sequence, base pairs with the 3' end of the 16S rRNA. This positions the 30S ribosomal subunit in relation to the initiation codon.  As the large ribosomal subunit joins the complex, GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1. A domain of the large ribosomal subunit serves as GAP (GTPase activating protein) for IF-2.  Once the two ribosomal subunits come together, the mRNA is threaded through a curved channel that wraps around the "neck" region of the small subunit.

Elongation requires participation of elongation factors • EF-Tu (also called EF1A) • EF-Ts (EF1B) • EF-G (EF2) EF-Tu & EF-G are small GTP-binding proteins. The sequence of events follows.

EF-Tu-GTP binds and delivers an aminoacyl-tRNA to the A site on the ribosome. EF-Tu recognizes & binds all aminoacyl-tRNAs with approximately the same affinity, when each tRNA is bonded to the correct (cognate) amino acid.

=EF-1

Proper reading of the anticodon is the second important quality control step ensuring accurate protein synthesis

Elongation factors Introduce a two-step “Kinetic proofreading”

A second elongation factor EF-G or EF-2, drives the translocation of the ribosome along the mRNA

Together GTP hydrolysis by EF-1 and EF-2 help drive protein synthesis forward

EF-Tu-GTP

ribosome (GAP) Pi EF-Tu-GDP

The change in ribosomal conformational associated with formation of a correct codon-anticodon complex leads to altered positions of active site residues in the bound EF-Tu, with activation of EF-Tu GTPase activity. The ribosome thus functions as GAP for EF-Tu.

As GTP on EF-Tu is hydrolyzed to GDP + Pi , EF-Tu undergoes a large conformational change & dissociates from the complex. The tRNA conformation relaxes, and the acceptor stem is repositioned to promote peptide bond formation. This process is called accommodation.

EF-Tu colored red

It includes rotation of the single-stranded 3' end of the acceptor stem of the A-site tRNA around an axis that bisects the peptidyl transferase center of the ribosomal large subunit. This positions the 3' end with its attached amino acid in the active site, near the 3' end of the P-site tRNA, & adjacent to the mouth of the tunnel through which nascent polypeptides exit the ribosome.

For images depicting the proposed rotational movement, see Fig. 5B in website of A. E. Yonath.

EF-Tu-GTP* GDP

EF-Ts functions as GEF to reactivate EF-Tu.

EF-Ts (GEF)

ribosome (GAP)

GTP

Pi EF-Tu-GDP**

*EF-Tu-GTP (conformation 1) binds & delivers aa-tRNA to A site on ribosome. **EF-Tu-GDP (conformation 2) dissociates from complex.

Interaction with EF-Ts causes EF-Tu to release GDP. Upon dissociation of EF-Ts, EF-Tu binds GTP, which is present in the cytosol at higher concentration than GDP.

Transpeptidation (peptide bond formation) involves acid/base catalysis by a universally conserved adenosine of the 23S rRNA of the large ribosomal subunit. The 23S rRNA may be considered a "ribozyme.“ The amino N of the amino acid linked to the 2' or 3' OH of the terminal adenosine of tRNA in the A site reacts with the carbonyl C of the amino acid (with attached nascent polypeptide) linked to the tRNA in the P site.

tRNA

P site

O

O O

P

A site

tRNA

O CH2

O−

H H O

O

P O

O CH2 −

H H O

C R

O

Adenine H H OH

O C

HC

R

:NH2

NH

O

H

O

H OH

O

HC

Adenine

C HC

R

NH3+

A ring N of the catalytic adenosine may promote the reaction by extracting a H+ from the attacking amino N. This H+ is then donated to the hydroxyl of the tRNA in the P site, as the ester linkage is cleaved.

The nascent polypeptide, one residue longer, is now linked to the A-site tRNA. Translocation has already partly occurred, because of the earlier rotation of the 3' end of the A-site tRNA toward the P-site, prior to catalysis.

tRNA

P site

O

O O

P O

A site

tRNA

O CH2 −

H H

OH

O

Adenine H

O

P O

O CH2 −

H H

H OH O

O

C R

NH O

C HC

R

NH

O

C HC

H H OH

O

HC

Adenine

R

NH3+

Peptide bond formation is catalyzed by the large subunit rRNA

Peptide bond formation is catalyzed by the large subunit rRNA

tRNA grey, EF-Tu red, EF-G blue

The unloaded tRNA in the P site will shift to an exit (E) site during translocation. Translocation of the ribosome relative to mRNA involves the GTP-binding protein EF-G.

large subunit

tRNA

EF-G

small subunit

mRNA location

Figure provided by Dr. J. Frank, Wadsworth Center. The tRNA with attached nascent polypeptide is pushed from the A site to the P site. Unloaded tRNA that was in the P site shifts to an exit site. Since tRNAs are linked to mRNA by codon-anticodon base pairing, the mRNA would move relative to the ribosome.

Additionally, it has been postulated that translocation is spontaneous after peptide bond formation because: • the deacylated tRNA in the P site has a higher affinity for the E site, & • the peptidyl-tRNA in the A site has a higher affinity for the P site. Interaction with the ribosome, which acts as GAP (GTPase activating protein) for EF-G, causes EF-G to hydrolyze its bound GTP to GDP + Pi. EF-G-GDP then dissociates from the ribosome. A domain of EF-G functions as its own GEF (guanine nucleotide exchange factor) to regenerate EF-G-GTP.

Chain termination requires release factors RF-1, RF-2, & RF-3. RF-3 is a small GTP-binding protein.  RF-1 & RF-2 recognize & bind to STOP codons. One or the other binds when a stop codon is reached.  RF-3-GTP facilitates binding of RF-1 or RF-2 to the ribosome.

Termination of translation is triggered by stop codons

Release factor enters the A site and triggers hydrolysis the peptidyl-tRNA bond leading to release of the protein.

Release of the protein causes the disassociation of the ribosome into its constituent subunits.

Ribosome Assembly The proteins of each ribosomal subunit are organized around rRNA molecules

16S rRNA

23S rRNA secondary structure