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fMet

C) 70S INITIATION COMPLEX

A) The small subunit and the mRNA bind to each other at the Shine-Dalgarno sequence. The start codon, AUG, is just downstream of this site. B) The initiator tRNA becomes tagged with fMet and binds to the AUG codon on the mRNA. C) The large ribosomal subunit joins the small subunit and accommodates the tRNA at the P-site.

Termination of Protein Synthesis Requires Release Factors 213

FIGURE 8.15 Overview of the Elongation Cycle on the Ribosome

After the first Met has been added, the incoming charged tRNA first occupies the A-site. The peptide bond is formed between the amino acid at the A-site and the growing polypeptide chain in the P-site. The uncharged tRNA exits the ribosome.

FIGURE 8.15 Overview of the Elongation Cycle on the Ribosome

After the first Met has been added, the incoming charged tRNA first occupies the A-site. The peptide bond is formed between the amino acid at the A-site and the growing polypeptide chain in the P-site. The uncharged tRNA exits the ribosome.

Uncharged tRNA

Phe Polypeptide Charged tRNA

chain

Only two tRNA molecules can occupy the ribosome at any instant.

After peptide bond formation the tRNA carrying the growing polypeptide chain moves sideways between sites on the ribosome.

The empty tRNA leaves the ribosome and a new, charged tRNA enters.

The stop codon is read by a protein, the release factor, not by a tRNA.

site, the P (peptide) site and the E (exit) site. However, only two charged tRNA molecules can be accommodated on the ribosome at any given instant (Fig. 8.15).

The fMet initiator tRNA starts out in the P-site. Another tRNA, carrying the next amino acid, arrives and enters the A-site. The fMet is cut loose from its tRNA and bonded to amino acid # 2, instead. So tRNA #2 now carries two linked amino acids, the beginnings of a growing polypeptide chain. (The enzyme activity that joins two amino acids together is referred to as the peptidyl transferase activity, as the growing peptide chain is transferred from the tRNA carrying it at each step.) After peptide bond formation, the two tRNAs are tilted relative to the A- and P-sites (Fig. 8.16). The tRNA carrying the growing polypeptide chain now occupies part of the A-site on the 30S subunit but part of the P-site on the 50S subunit. This is probably due to movement of the 50S subunit relative to the 30S subunit. The next step is translocation, in which the mRNA moves one codon sideways relative to the ribosome (Fig. 8.16). This moves the two tRNAs into the P- and E-sites, leaving the A-site empty.

When the next charged tRNA arrives, carrying the third amino acid, it enters the vacant A-site. This triggers release of the tRNA from the E-site. The A- and E-sites cannot be simultaneously occupied. As the peptide chain continues to grow, it is constantly cut off from the tRNA holding it and joined instead to the newest amino acid to be brought by its tRNA into the A-site, hence the name "acceptor" site.

Elongation requires two elongation factors, both of which use energy in the form of GTP. EF-T actually consists of a pair of proteins, EF-Tu and EF-Ts. Incoming charged tRNA is delivered to the ribosome and installed into the A-site by elongation factor EF-Tu. This requires energy from the hydrolysis of GTP. EF-Ts is responsible for exchanging the GDP left bound to EF-Tu for a fresh GTP. The second elongation factor, EF-G, oversees the translocation step (Fig. 8.16).

Termination of Protein Synthesis Requires Release Factors

Eventually the ribosome reaches the end of the message. This is marked by one of three possible stop codons, UGA, UAG, and UAA. As no tRNA exists to read these three codons, the polypeptide chain can no longer grow. Instead, proteins known

E (exit) site Site on the ribosome that a tRNA occupies just before leaving the ribosome elongation factors Proteins that are required for the elongation of a growing polypeptide chain P (peptide) site Binding site on the ribosome for the tRNA that is holding the growing polypeptide chain translocation a) Transport of a newly made protein across a membrane by means of a translocase;b) Sideways movement of the ribosome on mRNA during translation and c) Removal of a segment of DNA from a chromosome and its reinsertion in a different place

A) Acceptance of a new t-RNA

AUG mRNA

AUG mRNA

B) TRANSLOCATION OF t-RNA

FIGURE 8.16 Elongation Factors and Site Occupation

A) The EF-T factor is important in allowing a new charged tRNA to occupy the A-site. B) The EF-G factor is important in translocation of the tRNAs from the A- and P-sites, to the P- and E-sites, respectively. Note that during translocation the transfer RNA temporarily binds "diagonally" across two sites.

B) TRANSLOCATION OF t-RNA

FIGURE 8.16 Elongation Factors and Site Occupation

A) The EF-T factor is important in allowing a new charged tRNA to occupy the A-site. B) The EF-G factor is important in translocation of the tRNAs from the A- and P-sites, to the P- and E-sites, respectively. Note that during translocation the transfer RNA temporarily binds "diagonally" across two sites.

fMet|

as release factors (RF) read the stop signals (Fig. 8.17). RF1 recognizes UAA or UAG and RF2 recognizes UAA or UGA. The completed polypeptide chain is now released from the last tRNA. This is actually done by the peptidyl transferase. Binding of the release factor activates the peptidyl transferase which hydrolyzes the bond between the finished polypeptide chain and the tRNA in the P-site. The polypeptide chain, the tRNA and the mRNA now leave the ribosome, which dissociates into separate subunits. Two further factors aid in dissociation: RF3 releases RF1 or RF2 from the ribosome and ribosome recycling factor (RRF) dissociates the large and small subunits.

Messenger RNA is long enough for several ribosomes to translate it simultaneously.

Several Ribosomes Usually Read the Same Message at Once

Once the first ribosome has begun to move, another can associate with the same messenger RNA and travel along behind. In practice, several ribosomes will move along the same mRNA about a hundred bases apart (Fig. 8.18). An mRNA with several attached ribosomes is called a polysome (short for polyribosome).

Electron microscope observations have suggested that the polysomes of eukary-otic cells are circular (Fig. 8.19). Apparently, the 3'-end of the mRNA is attached to the 5'-end by protein-protein contact between the poly(A) binding protein (attached to the 3'-poly(A) tail) and the eukaryotic initiation factor, IF4 (attached to the cap at the 5'-end). In prokaryotic cells, such circularization cannot occur as the 3'-end of the mRNA is still being elongated by RNA polymerase while ribosomes have begun translating from the 5'-end.

polysome Group of ribosomes bound to and translating the same mRNA

release factor Protein that recognizes a stop codon and brings about the release of a finished polypeptide chain from the ribosome ribosome recycling factor (RRF) Protein that dissociates the ribosomal subunits after a polypeptide chain has been finished and released

Bacterial Messenger RNA Can Code for Several Proteins 215

FIGURE 8.17 Termination and Release of Finished Polypeptide

After the ribosome has added the final amino acid, release factors, RF1 and RF2, recognize the stop codon and cause the ribosome complex to dissociate.

Last translated codon

Last translated codon

Stop codon

Stop codon

RELEASE FACTORS RF1 & RF2

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