In bacteria, there is no barrier between the cell’s DNA and the cytoplasm, which contains the ribosomal subunits and all of the other components involved in polypeptide synthesis.Newly synthesized RNAs are released directly into the cytoplasm, where they can begin to interact with ribosomes. In fact, because the DNA is located in the cytoplasm in bacteria, the process of protein synthesis (translation) can begin before mRNA synthesis (transcription) is complete.
We will walk through the process of protein synthesis, but at each step we will leave out the various accessory factors involved in regulating the process and coupling it to the thermodynamically favorable reactions that make it possible. These can be important if you want to re-engineer or manipulate the translation system, but (we think) are unnecessary details that obscure a basic understanding. Here we will remind you of two recurring themes. The first is the need to recognize that all of the components needed to synthesize a new polypeptide (except the mRNA) are already present in the cell; another example of biological continuity. The second is that all of the interactions we will be describing are based on stochastic, thermally driven movements. For example, when considering the addition of an amino acid to a tRNA, random motions have to bring the correct amino acid and the correct tRNA to their binding sites on the appropriate amino acyl tRNA synthetase, and then bring the correct amino acid charged tRNA to the ribosome. Generally, many unproductive collisions occur before a productive (correct) one, since there are more than 20 different amino acid/tRNA molecules bouncing around in the cytoplasm. The stochastic aspects of the peptide synthesis process are rarely illustrated.
The first step in polypeptide synthesis is the synthesis of the specific mRNA that encodes the polypeptide. (1) The mRNA contains a sequence237 that mediates its binding to the small ribosomal subunit. This sequence is located near the 5’ end of the mRNA. (2) the mRNA-small ribosome subunit complex now interacts with and binds to a complex containing an initiator (start) amino acid:tRNA. In both bacteria and eukaryotes the start codon is generally an AUG codon and inserts the amino acid methionine (although other, non-AUG start codons are possible)238. This interaction 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 form a functional complex with a large ribosomal subunit to form the functional mRNA:ribosome complex. (4) Catalyzed by amino acid tRNA synthetases, charged amino acyl tRNAs will be present and can interact with the mRNA:ribosome complex to generate a polypeptide. Based on the mRNA sequence and the reading frame defined by the start codon, amino acids will be added sequentially. With each new amino acid added, the ribosome moves along the mRNA (or the mRNA moves through the ribosome). An important point, that we will return to when we consider the folding of polypeptides into their final structures, is that the newly synthesized polypeptide is threaded through a molecular tunnel within the ribosome. Only after the N-terminal end of the polypeptide begins to emerge from this tunnel can it begin to fold. (5) The process of polypeptide polymerization continues until the ribosome reaches a stop codon, that is a UGA, UAA or UAG239. Since there are no tRNAs for these codon, the ribosome pauses, waiting for a charged tRNA that will never arrive. Instead, a polypeptide known as release factor, which has 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.
When associated with the ribosome, the mRNA is protected against interaction with proteins that could degrade it (ribonucleases), that is, break it down into nucleotides. Upon its release the mRNA may interact with a new small ribosomesubunit, 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 particular polypeptides, the relative probabilities of these two events (new translation or RNA degradation) will be skewed in favor of degradation. Typically RNA stability is regulated by the bonding of specific proteins to nucleotide sequences within the mRNA. The relationship between mRNA synthesis and degradation will determine the half-life of a population of mRNA molecules within the cell, the steady state concentration of the mRNA in the cell, and indirectly, the level of polypeptide present.