The initiation process involves the large and small units of ribosomes. Ribosomes are megadalton molecules that are composed of ribosomal RNA (rRNA) and ribosomal proteins. The ribosomes direct protein synthesis by translating mRNA into a polypeptide chains. At physiological magnesium concentrations the ribosomes dissociate spontaneously into small 40s subunits and large 60s subunits of ribosomes. The large subunits contain the peptidyl transferase centre (catalyses the formation of the peptide bond), and the binding factor centre. The small subunits contain the decoding centre which the mRNA enters and exits through. Ribosomes contain three tRNA binding sites; ‘A site’ is the binding site for the aminoacylated tRNA, ‘P site’ is the binding site for the peptidyl-tRNA and ‘E site’ is the binding site for the uncharged tRNA. See figure 1 for the structure of a ribosome.
The first step in the initiation process is the formation of the pre-initiation complex consisting of the small ribosomal subunit, the special initiating tRNA charged with methionine (Met-tRNA), eukaryotic initiation factor (eIF2) and guanosine 5’-triphosphate (GTP). This pre-initiation complex then binds to the 5’ end of the mRNA and this step requires a cap binding complex (eIF4F) and eIF3. According to Hames and Hooper (2004) the efficiency of the initiation is influenced by the presence of a poly(A) tail at the 3’ end of the mRNA, probably via the ploy(A) binding protein that is bound to the tail, implying that the mRNA may bend back on itself to allow this interaction to occur. The complex then moves along the mRNA until it reaches the initiation codon (AUG) in a 5’ to 3’ prime direction. This initiation complex is usually recognised because it contains a short sequence called the Kozak consensus (5’-ACCAUGG-3’). Once the complex is positioned over the initiating codon the large ribosomal subunits bind to form an initiating complex, this binding requires energy which is produced by the hydrolysis of GTP. This initiation complex then occupies the peptidyl site (P-site) of the ribosome.
The elongation process of translations begins with the insertion of an aminoacyl-tRNA into the empty A-site on the ribosome. This aminoacyl-tRNA is delivered to the A-site by an elongation factor (EF-Tu). Since now there is a Met-tRNA in the P-site and aminoacyl-tRNA in the A-site a peptide bond can be produced involving a reaction which is catalysed by peptidyl transferase. This reaction causes the activated formylmethionine unit of the Met-tRNA to be transferred into the amino group of the aminoacyl-tRNA to form a dipeptidyl-tRNA. This therefore results in an uncharged tRNA occupying the P-site and a dipeptidyl-tRNA occupying the A-site, following the formation of a peptide bond. Then translocation occurs where the uncharged tRNA leaves the P-site, the peptidyl-tRNA moves from the A-site to the P-site and the mRNA moves a distance of three nucleotides. This results in the next codon been positioned for reading by the aminoacyl-tRNA.
Soon after the emergence of the amino-terminal signal recognition particle (SRP) from the ribosome the signal recognition particles bind tightly to these ribosomes. This causes the elongation of the polypeptide chain to be slowed down whilst the SRP is bound. This SRP-ribosome complex then diffuses to the endoplasmic reticulum membrane where the SRP binds to the SRP receptor. This therefore delivers the ribosome to the translocating areas and threads the polypeptide chain across the endoplasmic reticulum membrane. The SRP is then released from the receptor and binds to another signal sequence.
Termination of translation occurs because normally aminoacyl-tRNA does not bind to the A-site of a ribosome if the codon is UAA, UGA or UAG. These stop signals are not normally recognised by normal cells but are recognised by protein release factors. There are two types of release factors; RF1 (recognises UAA or UAG) and RF2 (recognises UAA or UGA). The binding of one of these release factors to a terminal codon in the A-site activates the peptidyl transferase so that the bond between the polypeptide and the tRNA in the P-site is hydrolysed. This causes the polypeptide chain to leave the ribosome which is followed by the tRNA and mRNA.
Hames, B.D. and Hooper, N.M. (2004) Instant Notes on Biochemistry, 2nd ed. Garland Science / BIOS Scientific Publishers, Oxon. Pp228.
Lodish, H., Baltimore, D., Berk, A., Zipursky, S.L., Matsudaira, P. and Darnell, J. (1995) Molecular Cell Biology, 3rd ed. Scientific American Books, New York. pp 119