Student Theses and Dissertations
Date of Award
1969
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Vincent Allfrey
Keywords
messenger RNA, codon translation, protein synthesis, Escherichia coli, codon composition, translation rate
Abstract
The order of addition of amino acids to a growing protein is determined by the codon sequence of a messenger RNA molecule. This translation process was studied in vitro with a cell-free protein synthesis system derived from Escherichia coli. The rate of protein synthesis was proportional to the amount of messenger RNA added to the system. However, it was observed that different messenger RNA's were not equally effective in promoting protein synthesis. Experiments were conducted to determine why the rate of protein synthesis depends on the type of messenger RNA. These experiments employed a nucleotide analogue, 5'-guanylyl-β,γ-methylenediphosphonate, which was shown to be a non-competitive inhibitor of protein synthesis. The presence of this inhibitor increases the time required to hydrolyze a guanosine-5'-triphosphate molecule. Since this hydrolysis is one of the sequential steps which occur each time a codon is translated, the effect of the inhibitor is to increase the time it takes to translate each codon. It was shown that the effectiveness of the inhibitor in reducing the rate of protein synthesis varied with the type of messenger RNA added to the system. Through the use of messenger RNA's whose coding properties were well characterized, it was possible to demonstrate that the inhibitor also has a differential effect on different cistrons within the same messenger RNA, and on different codons within the same cistron. Since the inhibitor probably extends the time it takes to translate each codon by a uniform amount of time, it was concluded that the differential effectiveness of the inhibitor was caused by intrinsic differences in the time it takes to translate each type of codon. Thus, different types of codons are translated at different rates. Each messenger RNA can be characterized as a collection of codons, with some types of codons occurring often and others rarely. Thus, the overall translation rate depends on the "codon composition" of the messenger RNA; and this is why the rate of protein synthesis was observed to depend on the type of messenger RNA added to the system. The significance of this conclusion is that the substitution of one codon for another in a messenger RNA can affect the rate at which that messenger RNA is translated. In particular, most amino acids can be specified by a number of different "synonym" codons. Though synonyms specify the same amino acid, the rate of translation probably varies from one synonym to another. Through the mechanism of natural selection, each amino acid in a protein could come to be specified by either a "fast" or a "slow" synonym codon. Thus, a gene might influence the amount of a protein synthesized, in addition to specifying the protein's amino acid sequence.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Kramer, Fred Russell, "Factors Affecting Translation of Messenger RNA's In Vitro: Use of a GTP Analogue to Investigate Rates of Polypeptide Chain Elongation" (1969). Student Theses and Dissertations. 564.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/564
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy