8.5: The translation (polypeptide synthesis) cycle

As we walk through the process of protein synthesis, keep in mind that at each step there are accessory factors involved in regulating the process and coupling it to the thermodynamically favorable reactions that make it possible. While important to consider if you want to re-engineer or manipulate the translation system, we think they obscure an understanding of the underlying process and are best considered in more advanced courses. It is important to recognize that mRNA-directed polypeptide synthesis (translation) occurs only because all the components needed already exist in the cell. The interactions between these components are based on stochastic, thermally driven collisions. For example, consider the addition of an amino acid to a tRNA, the formation of an amino acyl-tRNA or aa-tRNA. Random motions bring the correct amino acid and the correct tRNA to their binding sites on the appropriate amino acyl tRNA synthetase. Once the aa-tRNA is formed, (usually) only the correct amino acid charged tRNA will bind productively to the ribosome-mRNA-nascent polypeptide complex. If the wrong amino acid is inserted, the ribosome stalls and catalyzes its removal.^{(footnote 343)} Generally, many unproductive collisions occur before a productive (correct) one occurs, since there are more than 22 different amino acid/tRNA molecules bouncing around in the cytoplasm. The stochastic and error correcting aspects of the peptide synthesis process are, however, rarely illustrated.

The first step in polypeptide synthesis is the synthesis of the mRNA (←) that encodes the polypeptide. That mRNA (1) contains a sequence, located near the 5’ end of the mRNA, that mediates its productive binding to the small ribosomal subunit.^[334] (2) The mRNA-small ribosome subunit complex now interacts with and binds to a complex containing an initiator (start) amino acid:tRNA. In bacteria, archaea, and eukaryotes the start codon is generally an AUG codon and encodes the amino acid methionine, although other, non-AUG start codons are possible.^[335] This start codon-tRNA complex defines the beginning of the polypeptide as well as the coding region’s reading frame. (3) The met-tRNA:mRNA:small ribosome subunit complex can now interact with a large ribosomal subunit to form the functional mRNA:ribosome complex. (4) Charged amino acyl tRNAs can interact with the mRNA:ribosome complex. Based on the mRNA sequence and its start codon-defined reading frame, amino acids will be added sequentially. With each new amino acid added, the ribosome moves along the mRNA. An important point, that we will return to when we consider the folding of polypeptides into their final three-dimensional shapes is that the newly synthesized polypeptide is threaded through a molecular tunnel within the ribosome. Only after the N-terminal end of the polypeptide emerges from this tunnel does the nascent polypeptide begin to fold. (5) The process of polypeptide polymerization continues until the ribosome reaches a stop codon, that is a UGA, UAA or UAG.^[336] Since there are no tRNAs that recognize these codons, the ribosome pauses, waiting for a charged tRNA that will never arrive. Instead, a polypeptide known as release factor, with a shape something like a tRNA (→), binds to the polypeptide:mRNA:ribosome complex instead. (6) This leads to the release of the polypeptide, the disassembly of the ribosome into small and large subunits, and the release of the mRNA.^[337]
When associated with the ribosome, the mRNA is protected against interactions with proteins (ribonucleases) that could catalyze its degradation. Upon its release from the ribosome, an mRNA may interact with a new small ribosomal subunit, and begin the process of polypeptide synthesis again or it may interact with a ribonuclease and be degraded. Where it is important to limit the synthesis of a particular polypeptide, the relative probabilities of these two events, new translation versus RNA degradation, will be skewed in favor of degradation. An RNA's stability is regulated by the binding of specific proteins to nucleotide sequences within the mRNA. The relationship between mRNA synthesis and degradation will determine the half-life of the population of mRNA molecules, the steady state concentration of the mRNA in the cell, and indirectly, the level of the encoded polypeptide present.

In eukaryotes, RNA synthesis and processing occur primarily in the nucleus, while polypeptide synthesis occurs in the cytoplasm. In contrast, in bacteria and archaea, there is no barrier between the cell’s DNA and the cytoplasm, which contains the ribosomal subunits together with the other components involved in polypeptide synthesis. The newly synthesized 5' end of an mRNAs is deposited directly into the cytoplasm, where they can interact with ribosomes. In fact, the process of protein synthesis (translation) can begin before mRNA synthesis (transcription) is complete; the two processes can interact in interesting ways.^[338] Similar interactions can occur in eukaryotic mitochondria and chloroplasts, not surprising as they seem to be derived (evolutionarily) from bacteria.

---