(63)
13. Kort beskrive elongeringsfasen ud fra følgende:
Stryer, s.829-835
Devlin, s.245-251, fig. 6.8
 

Prokaryote celler:

50S ribosomal subunit samles med 30S (= 70S ribosom)

When the fMet-tRNA recognises the AUG of mRNA and binds to it, the 50S ribosomal subunit contacts the 30S subunit.

The 50S ribosomal subunit has three sites: A, P and E site.

When the first methionine-tRNA comes in, it binds to the P site. An aminoacyl (charged) tRNA, whose anticodon is complementary to the codon following the initiation AUG, takes the A site. The stage is set for formation of a peptide bond: the formylmethionine linked to the initiator tRNA will be transferred to the amino group of the amino-acid in the A-site. This transfer takes place at a special site of the peptidyltransferase enzyme.

After the formation of the peptide-bond, the uncharged CCA-region of the methionine tRNA is in the E site, while the CCA-region of the tRNA containing the growing polypeptide chain is located in the P-site, while their anti-codons are still in, respectively, the P and A-site of the small subunit.  

The polypeptide chain remains in the P-site of the large subunit, presumably growing in the channel that opens on the other side of the ribosome.
 

 
Elongeringsfaktorer
Stryer, s.834 - fig.29.28
Stryer, s.835 - fig.20.30
 

The process of elongation begins when the aminoacyl-tRNA complementary to the second codon is inserted into the A-site. The aminoacyl t-RNA doesn’t simply leave the aminoacyl t-RNA synthetase and diffuse to the A-site, rather it is delivered to the ribosome by an elongation factor – EF-Tu.

EF-Tu binds to the aminoacyl-tRNA only when bound to GTP. It protects the fragile ester bond between the adenylate-ribose and the carboxyl group of the amino acid. When the appropriate interaction is formed between the aminoacyl-tRNA anti-codon and the mRNA codon, the GTP is hydrolysed and EF-Tu releases tRNA. If the perfect bond is not formed, there is no hydrolysis and the aminoacyl tRNA is not transferred to the ribosome. 

A second factor, EF-Ts helps EF-Tu release the GDP and bind new GTP so it is capable of binding and delivering a new aminoacyl tRNA.  

EF-G is the prokaryotic enzyme that catalyses translocation of the uncharged tRNA from the P site to the E site of the smaller subunit, and the translocation of the polypeptidyl tRNA from the A to the P site again on the smaller subunit. A protein domain mimics the anticodon stem of tRNA.

GTP is used in the process, and the A-site is left completely empty.
 

Korrekt (cognate) ladet tRNA (i A-sitet)

Two enzymes play a crucial role:

Aminoacyl t-RNA synthetase is the enzyme that has to incorporate the correct amino acid to the CCA – acceptor stem of the tRNA. It has several structural domains that are used to find the right amino acid to the right tRNA.

It can determine if the right amino acid has been found by its bulk or lack of it, the positive or negative side chains. In the case when two amino acids are structurally similar but chemically different, it uses its catalytic site, which contains a jon which binds to one group but not to the other. If the amino acids are chemically similar, but have different structure, then a proofreading is required. For that purpose, synthetase has an extra editing site that is hydrolytic and cleaves amino acids that are smaller then the correct one, while the catalytic site is hydrophobe and cleaves amino acids that are too big.

The synthetase also checks if it has the right tRNA, by checking its anticodon sequence, its acceptor stem, the variable region and sometimes, the so-called micro-helices, which contain about 1/3 of all the sequences of an tRNA molecule. 

EF-Tu – the enzyme that brings the aminoacyl t-RNA to the ribosome after it has been released from the synthetase. It is a G-protein that binds the tRNA in its GTP form and disassociates from it if the anticodon triplet forms a complementary bond to the mRNA codon, since only then the GTP disassociates to GDP and Pi. If the codon-anticodon interactions are not complementary, then hydrolysis of GTP does not occur and the aminoacyl-tRNA is not released in the A-site.
 

Eukaryote celler:
Devlin, s.250
Stryer, s.837-838
 

60S ribosomal subunit samles med 40S (=80S ribosom)

When the AUG codon is located by the scanning complex of 40S subunit, Met-tRNAi and GTP, the large subunit is bound with the help of many initiation factors. GTP is then released from the complex.

The principle of moving the tRNAs from A to P and E site of the big subunit in eukaryotes is basically the same as what goes on in prokaryotes.
 

 
Elongeringsfaktorer (EF-1alfa og EF-2)

EF - 1α in eukaryotes has the same function as EF-Tu in prokaryotes.

EF - 1βγ is the equivalent of EF – Ts.

EF - 2 is the eukaryotic enzyme that catalyses translocation and it pretty much has the same function as EF-G in prokaryotes. (not sure if it also mimics the anticodon stem as EF-G does)

EF - 1α and EF-2 are G-proteins, bound to GTP they have a high affinity for aminoacyl t-RNA and ribosome, while bound to GDP they disassociate from them more easily. Same goes for the prokaryotic equivalents.

Korrekt (cognate) ladet tRNA (i A-sitet)

Again, completely the same principle as in prokaryote, just that EF-Tu is EF-1α.

Peptidyl transferase aktivitet (peptid binding)

Peptydiltransferase is an enzyme that transfers the growing peptide (or in the first step the initiating methionine residue) from its carrier tRNA to the -amino group of the amino-acid residue of the aminoacyl t-RNA specified by the next codon. 

Whatever the length of the growing chain, peptide bond formation always occurs through attack of the α-amino group of the incoming aminoacyl-tRNA on the carbonyl group of the ester linkage of the peptidyl t-RNA to form a tetrahedral intermediate that collapses to form the peptide bond and the deacylated tRNA.

The transfer occurs in a ribosome site called the peptidyltransferase center.  

Peptydiltransferase has never been dissociated from the large subunit or characterised as a specific ribosomal protein. It is thought that the large subunit is a complex ribozymes in which peptide bond formation is a RNA-catalysed reaction.
 

Translokation
 

After the formation of the peptide-bond, the uncharged CCA-region of the methionine tRNA is in the E site, while the CCA-region of the tRNA containing the growing polypeptide chain is located in the P-site, while their anticodons are still in, respectively, the P and A-site of the small subunit. 

The mRNA must move by a distance of three nucleotides and the dipeptidyl-tRNA must be repositioned to permit another elongation cycle to begin. The repositioning happens on the small subunit because the respective areas of the tRNA on the large subunit have already been moved by peptidyltransferase.

This process is called translocation and is catalysed by EF2 in eukaryotes and EF-G in prokaryotes, enzymes also called translocases.  

A protein domain of EF-G mimics the anticodon stem of tRNA! In the process, the uncharged tRNA is moved out of the P-site of the small subunit into the E-site and completely disengages from the mRNA. (The entire uncharged tRNA is now moved to the E-site)

The dipeptidyl (later polypeptidyl) tRNA is moved from the A to the P site. (The entire polypeptidyl tRNA is now moved to the P-site)

 The A-site is completely vacant (both in the large and small subunit) and a new cycle can begin. 

During translocation, GTP is used and splits to GDP and Pi.
 

Frigivelse af brugte (deacetylerede) tRNA’er gennem E-sitet

The uncharged tRNAs are released from the mRNA in the process of translocation by translocases.

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