A ribosome translating a protein that is secreted into the endoplasmic reticulum
. tRNAs are colored dark blue.
Tertiary structure of tRNA. CCA tail
in yellow, Acceptor stem
in purple, Variable loop
in orange, D arm
in red, Anticodon arm
in blue with Anticodon
in black, T arm
The basic process of translation is the addition of three amino acids at a time to the end of the polypeptide being formed. This process takes place inside the ribosome. A ribosome is made up of two subunits, a small 40S subunit and a large 60S subunit. These subunits come together before translation of mRNA into a protein to provide a location for translation to be carried out and a polypeptide to be produced. The choice of amino acid type to be added is determined by the genetic code on the mRNA molecule. Each amino acid added is matched to a three nucleotide subsequence of the mRNA. For each such triplet possible, the corresponding amino acid is accepted. The successive amino acids added to the chain are matched to successive nucleotide triplets in the mRNA. In this way, the sequence of nucleotides in the template mRNA chain determines the sequence of amino acids in the generated polypeptide.
Addition of an amino acid occurs at the C-terminus of the peptide and thus translation is said to be amino-to-carboxyl directed.
The mRNA carries genetic information encoded as a DNA sequence from the chromosomes to the nucleolus. The ribonucleotides are "read" by translational machinery in a sequence of nucleotide triplets called codons. Each of those triplets codes for a specific amino acid.
The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multi-subunit structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins.
tRNAs are small noncoding RNA chains (75-90 nucleotides) that transport amino acids to the ribosome. tRNAs have a site for amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their cargo amino acid.
Aminoacyl tRNA synthetases (enzymes) catalyze the bonding between specific tRNAs and the amino acids that their anticodon sequences call for. The product of this reaction is an aminoacyl-tRNA. In prokaryotes, this aminoacyl-tRNA is carried to the ribosome by EF-Tu, where mRNA codons are matched through complementary base pairing to specific tRNA anticodons. Aminoacyl-tRNA synthetases that mispair tRNAs with the wrong amino acids can produce mischarged aminoacyl-tRNAs, which can result in inappropriate amino acids at the respective position in protein. This "mistranslation" of the genetic code naturally occurs at low levels in most organisms, but certain cellular environments cause an increase in permissive mRNA decoding, sometimes to the benefit of the cell.
The ribosome has three sites for tRNA to bind. They are the aminoacyl site (abbreviated A), the peptidyl site (abbreviated P) and the exit site (abbreviated E). With respect to the mRNA, the three sites are oriented 5’ to 3’ E-P-A, because ribosomes move toward the 3' end of mRNA. The A-site binds the incoming tRNA with the complementary codon on the mRNA. The P-site holds the tRNA with the growing polypeptide chain. The E-site holds the tRNA without its amino acid. When an aminoacyl-tRNA initially binds to its corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the tRNA in the A site and the amino acid of the charged tRNA in the P site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA in the P site, now without an amino acid, to the E site; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P site. The tRNA in the E site leaves and another aminoacyl-tRNA enters the A site to repeat the process.
After the new amino acid is added to the chain, and after the mRNA is released out of the nucleus and into the ribosome's core, the energy provided by the hydrolysis of a GTP bound to the translocase EF-G (in prokaryotes) and eEF-2 (in eukaryotes) moves the ribosome down one codon towards the 3' end. The energy required for translation of proteins is significant. For a protein containing n amino acids, the number of high-energy phosphate bonds required to translate it is 4n+1. The rate of translation varies; it is significantly higher in prokaryotic cells (up to 17-21 amino acid residues per second) than in eukaryotic cells (up to 6-9 amino acid residues per second).
Even though the ribosomes are usually considered accurate, processive machines, the translation process is subject to errors that can lead either to the synthesis of erroneous proteins or to the premature abandonment of translation. The rate of error in synthesizing proteins has been estimated to be between 1/105 and 1/103 misincorporated amino acids, depending on the experimental conditions. The rate of premature translation abandonment, instead, has been estimated to be of the order of magnitude of 10−4 events per translated codon.
The correct amino acid is covalently bonded to the correct transfer RNA (tRNA) by amino acyl transferases. The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, the tRNA is termed "charged". Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). In prokaryotes, initiation of protein synthesis involves the recognition of a purine-rich initiation sequence on the mRNA called the Shine-Dalgarno sequence. The Shine-Dalgarno sequence binds to a complementary pyrimidine-rich sequence on the 3' end of the 16S rRNA part of the 30S ribosomal subunit. The binding of these complementary sequences ensures that the 30S ribosomal subunit is bound to the mRNA and is aligned such that the initiation codon is placed in the 30S portion of the P-site. Once the mRNA and 30S subunit are properly bound, an initiation factor brings the initiator tRNA-amino acid complex, f-Met-tRNA, to the 30S P site. The initiation phase is completed once a 50S subunit joins the 30 subunit, forming an active 70S ribosome. Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA) on the mRNA. tRNA usually cannot recognize or bind to stop codons. Instead, the stop codon induces the binding of a release factor protein that prompts the disassembly of the entire ribosome/mRNA complex and the hydrolysis and the release of the polypeptide chain from the ribosome. Drugs or special sequence motifs on the mRNA can change the ribosomal structure so that near-cognate tRNAs are bound to the stop codon instead of the release factors. In such cases of 'translational readthrough', translation continues until the ribosome encounters the next stop codon.
The process of translation is highly regulated in prokaryotic and eukaryotic organisms. Regulation of translation can impact the global rate of protein synthesis which is closely coupled to the metabolic and proliferative state of a cell. In addition, recent work has revealed that genetic differences and their subsequent expression as mRNAs can also impact translation rate in an RNA-specific manner.