Translation (biology) - Wikipedia

Further information: Bacterial translation, Archaeal translation, and Eukaryotic translation

 

 

The basic process of protein production is the addition of one amino acid at a time to the end of a protein. This operation is performed by a ribosome.[2] A ribosome is made up of two subunits, a small subunit, and a large subunit. These subunits come together before the translation of mRNA into a protein to provide a location for translation to be carried out and a polypeptide to be produced.[3] The choice of amino acid type to add is determined by a messenger RNA (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 amino acid chain.[4] The addition of an amino acid occurs at the C-terminus of the peptide; thus, translation is said to be amine-to-carboxyl directed.[5]

The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosomes. 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.[citation needed]

The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multisubunit structure containing ribosomal RNA (rRNA) and proteins. It is the "factory" where amino acids are assembled into proteins.

Transfer RNAs (tRNAs) are small noncoding RNA chains (74–93 nucleotides) that transport amino acids to the ribosome. The repertoire of tRNA genes varies widely between species, with some bacteria having between 20 and 30 genes while complex eukaryotes could have thousands.[6] 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. 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". In bacteria, 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 the protein. This "mistranslation"[7] 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 two binding sites for tRNA. They are the aminoacyl site (abbreviated A), and the peptidyl site/ exit site (abbreviated P/E). Concerning 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/E-site holds the tRNA with the growing polypeptide chain. 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/E site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA to the P/E site, now without an amino acid; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P/E site and the uncharged tRNA leaves, and another aminoacyl-tRNA enters the A site to repeat the process.[8]

After the new amino acid is added to the chain, and after the tRNA is released out of the ribosome and into the cytosol, the energy provided by the hydrolysis of a GTP bound to the translocase EF-G (in bacteria) and a/eEF-2 (in eukaryotes and archaea) 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.[9] 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).[10]

Initiation and termination of translation

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Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). In bacteria and a minority of archaea, 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 30S subunit, forming an active 70S ribosome.[11] Termination of the polypeptide occurs when the A site of the ribosome is occupied by a stop codon (UAA, UAG, or UGA) on the mRNA, creating the primary structure of a protein. tRNA usually cannot recognize or bind to stop codons. Instead, the stop codon induces the binding of a release factor protein[12] (RF1 & RF2) that prompts the disassembly of the entire ribosome/mRNA complex by the hydrolysis of the polypeptide chain from the peptidyl transferase center [2] of the ribosome.[13] 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.[14]

Errors in translation

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Even though the ribosomes are usually considered accurate and 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, either because a tRNA couples to a wrong codon or because a tRNA is coupled to the wrong amino acid.[15] The rate of error in synthesizing proteins has been estimated to be between 1 in 105 and 1 in 103 misincorporated amino acids, depending on the experimental conditions.[16] The rate of premature translation abandonment, instead, has been estimated to be of the order of magnitude of 10−4 events per translated codon.[17][18]

Regulation

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The process of translation is highly regulated in both eukaryotic and prokaryotic 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.

To study this process, scientists have used a wide variety of methods such as structural biology, analytical chemistry (mass-spectrometry based), imaging of reporter mRNA translation (in which the translation of a mRNA is linked to an output, such as luminescence or fluorescence), and next-generation sequencing based methods.[19] Other methods such as toeprinting assay can also be used to determine the location of ribosomes of a particular mRNA in vitro, and footprints of other proteins regulating translation.

In particular, ribosome profiling, which is a powerful method,[20] enables researchers to take a snapshot of all the proteins being translated at a given time, showing which parts of the mRNA are being translated into proteins by ribosomes at a given time. This method is useful because it looks at all the mRNAs instead of using reporters that would typically look at one specific mRNA at a time. Ribosome profiling provides valuable insights into translation dynamics, revealing the complex interplay between gene sequence, mRNA structure, and translation regulation. For example, research utilizing this method has revealed that genetic differences and their subsequent expression as mRNAs can also impact translation rate in an RNA-specific manner.[21]

Expanding on this concept, a more recent development is single-cell ribosome profiling, a technique that allows us to study the translation process at the resolution of individual cells.[22] This is particularly significant as cells, even those of the same type, can exhibit considerable variability in their protein synthesis. Single-cell ribosome profiling has the potential to shed light on the heterogeneous nature of cells, leading to a more nuanced understanding of how translation regulation can impact cell behavior, metabolic state, and responsiveness to various stimuli or conditions.