In translation, all of the elements needed to synthesize a protein, from the mRNA to the tRNAs with amino acids, are brought together on the ribosomes (figure 1). The process occurs in five stages: initiation, elongation, termination, and protein folding and processing.
Initiation of Translation
In prokaryotic cells, the mRNA molecule leaves the DNA transcription site and is transported directly to ribosomes. Ribosomal sub units are specifically assembled in a way that forms sites to hold the mRNA and tRNAs. The ribosome thus recognizes these molecules and stabilizes reactions between them. The small subunit binds to the 5′ end of the mRNA and provides molecules to initiate translation. The large subunit holds the tRNAs and is actively involved in peptide bond formation by means of a specialized ribozyme, which is an RNA-based catalyst. The small subunit of the ribosome binds at a specific site on the mRNA and places the start codon (AUG) in correct alignment with the P site.
With the mRNA message in place on the assembled ribosome, the next step in translation involves entrance of tRNAs with their amino acids. The pool of cytoplasm around the region contains a complete array of tRNAs, previously charged by having the correct amino acid attached. The step in which the complementary tRNA meets with the mRNA code is guided by the two sites on the large subunit of the ribosome called the P site (left) and the A site (right). Think of these sites as recessed spaces tucked within the two sub units of the ribosome, with each site accommodating a tRNA. The ribosome also has an exit or E site where used tRNAs are released.
The Master Genetic Code: The Message in Messenger RNA
By convention, the master genetic code is represented by the mRNA codons and the amino acids they specify (figure 1). Except in a very few cases (the genes of mitochondria and chloroplasts, for ex ample), this code is universal, whether for prokaryotes, eukaryotes, or viruses. It is worth noting that once the mRNA codon is known, the original DNA sequence, the complementary tRNA code, and the types of amino acids in the protein are automatically known (figure 2). However, one cannot predict (backward) from protein structure what the exact mRNA codons are because of a factor called redundancy or degeneracy, meaning that a particular amino acid can be coded for by more than one codon.
Fig1. The genetic code: Codons of mRNA that specify a given amino acid. The master code for translation is provided by mRNA codons.
Fig2. Interpreting the DNA code. If the DNA sequence is known, the mRNA codon can be surmised. If a codon is known, the anticodon and, finally, the amino acid sequence can be determined. The reverse is not possible (determining the exact codon or anticodon from amino acid sequence) due to the redundancy of the code.
In figure 1 the mRNA codons and their corresponding amino acid specificities are given. Because there are 64 different triplet codes2 and only 20 different amino acids, some amino acids are represented by several codons. For example, leucine and serine can each be represented by any of six different triplets, but tryptophan and methionine are represented by a single codon. In such codons as leucine, only the first two nucleotides are required to encode the correct amino acid, and the third nucleotide does not change its sense. This property, called wobble, is thought to permit some level of variation or mutation without changing the message.
The Beginning of Protein Synthesis
When the correct tRNA enters the P site, hydrogen bonds will form between the codon (mRNA) and anticodon (tRNA). These bonds will stabilize the tRNA, and it will remain in place. If an incorrect tRNA enters, no bonding occurs between the codon and anticodon, and the tRNA will float out of the ribosome. The amino acid corresponding to the start codon (AUG) is formyl methionine (fMet, a modified amino acid), though in many cases it may not remain a part of the completed protein.
Continuation and Completion of Protein Synthesis: Elongation and Termination
Beginning with the start codon, the mRNA is read by the ribosome one codon (three nucleotides) at a time. The ribosome is mobile and moves along the mRNA. When two tRNAs, each carrying a single amino acid, are present in the ribosome (one in the A site and one in the P site), joining of the amino acids is carried out by a ribozyme (a catalytic RNA molecule) that is part of the large subunit of the ribosome. It forms a peptide bond between the amino acids on the adjacent tRNAs, and the polypeptide grows in length. Several steps of this process are outlined here:
● Initiation begins when a start codon (AUG) is encountered.
● Elongation starts with the filling of the A site by a second tRNA (process figure 3, step 1). The identity of this tRNA and its amino acid is dictated by the second mRNA codon.
● A peptide bond forms between the amino acids attached to the two adjacent tRNAs in the ribosome. The fMet is transferred from the first tRNA to amino acid 2, resulting in two coupled amino acids called a dipeptide (process figure 3, step 2).
● For the next step to proceed, some room must be made on the ribosome, and the next codon in sequence must be brought into position for reading. This process is accomplished by translocation, the enzyme-directed shifting of the ribosome to the next position on the mRNA strand, which causes the empty tRNA (tRNA 1) to be discharged from the ribosome (process figure 3, step 3) at the E site.
● This simultaneously shifts the tRNA holding the dipeptide into the P position. Site A is temporarily left empty. The tRNA that has been released is now free to drift away and become recharged with an amino acid for later additions to this or another protein.
● The stage is now set for the insertion of tRNA 3 at site A as directed by the third mRNA codon (process figure 9.18, step 4). Another peptide bond is formed (creating a tripeptide), the empty tRNA is released, and the ribosome translocates to the next codon (process figure 3, step 5).
● This action releases tRNA 2, shifts mRNA to the next position, moves tRNA 3 to position P, and opens position A for tRNA 4 (step 6). From this point on, peptide elongation proceeds re petitively by this same series of actions out to the end of the mRNA (steps 7 and 8).
Fig3. The events in protein synthesis.
The termination of protein synthesis is not simply a matter of reaching the last codon on mRNA. It is brought about by the presence of one of three specific codons. Termination codons—UAA, UAG, and UGA— are codons for which there is no corresponding tRNA. Termed stop codons, they carry a necessary message: Stop here. When this codon is reached, a special enzyme breaks the bond between the final tRNA and the finished polypeptide chain, releasing it from the ribosome.
Before newly made proteins can carry out their structural or enzymatic roles, they often require finishing touches. Even before the peptide chain is released from the ribosome, it begins folding upon itself to achieve its biologically active conformation. Other alterations, called posttranslational modifications, may be necessary. Some proteins must have the starting amino acid (formyl me thionine) clipped off; proteins destined to become complex enzymes have cofactors added; and some join with other completed proteins to form quaternary levels of structure.
The operation of transcription and translation is machinelike in its precision. Protein synthesis in bacteria is both efficient and rapid. At 37°C, 12 to 17 amino acids per second are added to a growing peptide chain. An average protein consisting of about 400 amino acids requires less than half a minute for complete synthesis. Further efficiency is gained when the translation of mRNA starts while transcription is still occurring (figure 4). A single mRNA is long enough to be fed through more than one ribosome simultaneously. This permits the synthesis of hundreds of protein molecules from the same mRNA transcript arrayed along a chain of ribosomes. This polyribosomal complex is indeed an assembly line for mass production of proteins. Protein synthesis consumes an enormous amount of energy. Approximately 1,200 ATPs or ATP equivalents are consumed for synthesis of an average-size protein.
Fig4. Speeding up the protein assembly line in bacteria. (a) The mRNA transcript encounters ribosomal parts immediately as it leaves the DNA. (b) The ribosomal factories assemble along the mRNA in a chain, each ribosome reading the message and translating it into protein. Many products will thus be well along the synthetic pathway before transcription has even terminated. (c) Photomicrograph of a polyribosomal complex in action. Note that the protein “tails” vary in length depending on the stage of translation (30,000×). (c) Steven McKnight and Oscar L Miller, Department of Biology, University of Virginia
