Elongation Rate And Its Modulation

13.1. Elongation Rates in Prokaryotes and Eukaryotes

During the elongation stage of translation the elongation cycle consisting of three main steps, namely aminoacyl-tRNA binding, transpeptidation and translocation (Fig. 9.1), repeats as many times as many sense codons are present in the coding sequence of a message. Thus, the cycle frequency directly determines the polypeptide elongation rate.

13.1.1. Transit Time

The time during which a growing nascent peptide remains attached to the translating ribosome, i.e., the time of its elongation plus termination, is called transit time. If the termination time is neglected, the number of amino acids in a given protein divided by its transit time is the average rate of elongation on a corresponding mRNA. Hence, the transit time determinations for proteins of known size may give an information about the elongation rate (for review, see Nielsen & McConkey, 1980).

To measure the transit time, usually a radioactive amino acid is given to a cell. The radioactivity will soon appear in polyribosomes in the form of growing nascent peptides attaches to ribosomes. The radioactivity of the polyribosome fraction of the cell will increase as the nascent peptides on ribosomes will be elongated by the labeled amino acids (Fig. 13.1). When all the growing peptides become fully labeled the plateau of radioactivity of the polyribosome fraction will be established. The time of reaching the plateau is the transit time.

In practice, it is often more convenient and accurate to determine the transit time from the kinetics of radioactivity incorporation into the ribosome-free fraction of the soluble (completed) proteins as compared with that of the total incorporation into polypeptides. The incorporation into soluble protein will increase first exponentially and then, after reaching the plateau in the polyribosome fraction, linearly at the expense of the release of fully labeled polypeptides. At the same time it is evident that the total incorporation of the radioactive amino acid into polypeptides should be linear almost from the beginning (provided the elongation rate is constant during the experiment). Since after reaching the radioactivity plateau in polyribosomes the increment of the radioactivity both in total peptides and in soluble protein fraction is determined by the same process of releasing labeled polypeptides from ribosomes the two linear plots should be parallel (Fig. 13.1, lower panel). The distance between them along the abscissa corresponds to the time required for the completion of the synthesis nascent peptides in polyribosomes, i.e., to half transit time. (A reminder should be that the average length of nascent peptides in a polyribosome is always half of the full length of completed polypeptides, i.e., at each given moment a polyribosome contains half-completed polypeptides on average).

13.1.2.Average Elongation Rate and Its Variations

From measuring the transit time, the rates of elongation of different polypeptides (average elongation cycle frequencies on different mRNAs) in both prokaryotic and eukaryotic cells, as well as in cell-free systems, were estimated. In Escherichia coli, up to 15 to 20 codons per second can be read out on some mRNAs at saturating substrate and factor concentrations (rich medium) at 37*C. The rate of 10 codons per second is more typical of the bacteria growing in a poorer medium. The same rate of about 10 codons per second can be achieved in a bacterial cell-free system under optimized conditions. Thus, the average time of the elongation cycle in bacterial systems varies usually from 0.05 sec to 0.1 sec at 37*C, an it is three times longer at 25*C.

The elongation rate in eukaryotic systems can reach 10 codons per second at 37*C, but usually it is lower and varies in a wide range due to the presence of control mechanisms regulating elongation. Typical variations of the time of the eukaryotic elongation cycle are from 0.1 sec to 0.5 sec.

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