Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Biosynthesis
Translation and General Requirements for Protein Synthesis in a Cell-Free System
Messenger RNA

Previously, it was noted that a pre-existing RNA molecule is required for the correct alignment of Amino Acids within a polypeptide chain. Until relatively recently, it was assumed that rRNA could serve as such a molecule. This assumption seemed to be supported by experiments on infecting E. coli Cells with phage DNA. It turned out that immediately after infection, the synthesis of normal cellular DNA ceases, and the intensive synthesis of phage DNA begins. Moreover, the entire protein-synthesizing apparatus of the cells was reprogrammed to synthesize phage Proteins exclusively. The COMPOSITION OF THE synthesized phage proteins differed from that of the bacterial proteins. It was suggested that phage infection presumably alters The base sequence in RNA, but this hypothesis was not confirmed either. According to several prominent scientists, the pre-existing RNA required to alter the type of synthesized protein must possess a high rate of turnover, meaning that such an RNA molecule must be synthesized and degraded at a rate sufficient to support this rapid turnover of its nucleotide composition. In fact, rRNA proved to be metabolically inactive and highly stable, making it obvious that it could not function as a template.

Data obtained in A number of laboratories (specifically, in S. Brenner's laboratory) indicated the possible presence in cells, associated with Ribosomes, of a short-lived RNA* termed informational RNA (iRNA). It is now designated as Messenger RNA (mRNA) because its role is to carry information from the DNA in The Nucleus (where it is synthesized by DNA-dependent RNA polymerase) to the Cytoplasm, where it binds to ribosomes and serves as the template on which Protein Synthesis takes place. This brilliant hypothesis was subsequently proven experimentally in M. Nirenberg's laboratory. When studying The Effect of various cellular RNA fractions on the protein-synthesizing capacity of ribosomes isolated from E. coli, it was established that some of them stimulated the incorporation of ¹⁴C-labeled amino acids into the synthesized polypeptide. The addition of a synthetic polynucleotide, specifically polyuridylic acid (poly-U), to the protein-synthesizing system led to the incorporation of a single amino acid—phenylalanine—into the synthesized protein molecule. Poly-U directed the synthesis of an unusual polypeptide, polyphenylalanine, in a Cell-free system. Thus, an artificially synthesized polyribonucleotide added to ribosome preparations containing the protein synthesis factors and Energy Sources known at the time triggered the synthesis of a specific, programmed polypeptide.

These experiments opened up the possibility of experimentally deciphering the entire Genetic Code by which information is transferred from RNA to the synthesized protein. The nucleotide sequence of RNA is translated into the specific Amino Acid Sequence of the synthesized polypeptide chain. M. Nirenberg's experiments also demonstrate that neither the ribosome nor ribosomal rRNA acts as the template upon which specific proteins are synthesized; rather, this role is performed by exogenously supplied messenger RNAs. Thus, DNA transfers information to RNA, which is synthesized in the nucleus and then enters the cytoplasm; here, RNA performs a template function for the synthesis of a specific protein molecule. The template hypothesis for proteins, as well as for other polymer molecules such as DNA and R.N.A. (see above), has now been confirmed. Its validity was proven in experiments ensuring the precise Replication of the Introduction/19.html">Primary Structure of polymer molecules. Unlike poorly controlled chemical synthesis, this synthesis was distinguished not only by high speed and Specificity, but also by the directional Nature of the process itself, strictly in accordance with the program encoded in the linear sequence of the template molecule.

* The possible existence of a rapidly turning-over RNA molecule in cells was first pointed out in the works of A.N. Belozersky and A.S. Spirin.