Translation (biology)
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Translation is the second process of protein biosynthesis (part of the overall process of gene expression).Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surrounds the mRNA. In translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide according to the rules specified by the genetic code. This is the process that converts an mRNA sequence into a chain of amino acids that form a protein. Translation is necessarily preceded by transcription. Translation proceeds in four phases: activation, initiation, elongation and termination (all describing the growth of the amino acid chain, or polypeptide that is the product of translation). In activation, the correct amino acid (AA) is joined to the correct transfer RNA (tRNA). While this is not technically a step in translation, it is required for translation to proceed. The AA 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, it is termed "charged". Initiation involves the small subunit of the ribosome binding to 5' end of mRNA with the help of initiation factors (IF), other proteins that assist the process. Elongation occurs when the next aminoacyl-tRNA (charged tRNA) in line binds to the ribosome along with GTP and an elongation factor. Termination of the polypeptide happens when the A site of the ribosome faces a stop (nonsense) codon (UAA, UAG, or UGA). When this happens, no tRNA can recognize it, but releasing factor can recognize nonsense codons and causes the release of the polypeptide chain. The capacity of disabling or inhibiting translation in protein biosynthesis is used by antibiotics such as: anisomycin, cycloheximide, chloramphenicol and tetracycline.
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[edit] Basic mechanisms
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. The ribosome and tRNA molecules translate this code to produce proteins. The ribosome is a multisubunit structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs are small noncoding RNA chains (74-93 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 synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids that their anticodons sequences call for. The product of this reaction is an aminoacyl-tRNA molecule. This aminoacyl-tRNA travels inside the ribosome, where mRNA codons are matched through complementary base pairing to specific tRNA anticodons. The amino acids that the tRNAs carry are then used to assemble a protein. 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. It is also the process whereby ribosomes use the sequence of codons in mRNA to produce a polypeptide with a particular sequence of amino acids.
[edit] Prokaryotic translation
[edit] Initiation
Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: the two ribosomal subunits, the mRNA to be translated, the first aminoacyl tRNA (the tRNA charged with the first amino acid), GTP (as a source of energy), and initiation factors which help the assembly of the initiation complex. Prokaryotic initiation results in the association of the small and large ribosomal subunits and binding of first aminoacyl tRNA (fmet-tRNA) through anticodon-codon base pairing with the initiation codon of mRNA.
The ribosome consists of three sites: the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fmet-tRNA, which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
Initiation of translation begins with the 50s and 30s ribosomal subunits dissociated. IF1 (initiation factor 1) blocks the A site to insure that the fMet-tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A site during initiation, while IF3 blocks the E site and prevents the two subunits from associating. IF-2 is a small GTPase which binds fmet-tRNA and helps its binding with the small ribosomal subunit. The 16s rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on mRNA (the Shine-Dalgarno sequence, 5-10 base pairs upstream of the start codon(AUG)) The Shine-Delgarno sequence is found only in prokaryotes. This helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon. IF-3 helps to position fmet-tRNA into the P site, such that fmet-tRNA interacts via base pairing with the mRNA initiation codon (AUG). Initiation ends as the large ribosomal subunit joins the complex causing the dissociation of initiation factors. Note that prokaryotes can differentiate between a normal AUG (coding for methionine) and an AUG initiation codon (coding for formylmethionine and indicating the start of a new translation process).
[edit] Elongation
Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit[1].
Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next aminoacid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation, is catalyzed by a ribozyme, peptidyltransferase, an activity intrinsic to the 23S rRNA in the 50s ribosomal subunit. Now, the A site has newly formed peptide, while the P site has an unloaded tRNA (tRNA with no amino acids). In the final stage of elongation, translocation, the ribosome moves 3 nucleotides towards the 3'end of mRNA. Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site. This process is catalyzed by elongation factor G (EF-G).
The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).
[edit] Termination
Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons). A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process. These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome.
[edit] Recycling
The post-termination complex formed by the end of the termination step consists of mRNA with the termination codon at the A-site, tRNAs and the ribosome. Ribosome recycling step is responsible for the disassembly of the post-termination ribosomal complex. Once the nascent protein is released in termination, Ribosome Recycling Factor and Elongation Factor G (EF-G) function to release mRNA and tRNAs from ribosomes and dissociate the 70s ribosomes into the 30s and 50s subunits. IF-3 also helps the ribosome-recycling process by converting transiently dissociated subunits into stable subunits by binding to the 30S subunits. This "recycles" the ribosomes for additional rounds of translation.
[edit] Polysomes
Translation is carried out by more than one ribosome simultaneously. Because of the relatively large size of ribosomes, they can only attach to sites on mRNA 35 nucleotides apart. The complex of one mRNA and a number of ribosomes is called a polysome or polyribosome.
[edit] Effect of antibiotics
Several antibiotics exert their action by targeting the translation process in bacteria. They exploit the differences between prokaryotic and eukaryotic translation mechanisms to selectively inhibit protein synthesis in bacteria without affecting the host. Examples include:
- Puromycin has a structure similar to the tyrosinyl aminoacyl-tRNA. Thus, it binds to the ribosomal A site and participates in peptide bond formation, producing peptidyl-puromycin. However, it does not engage in translocation and quickly dissociates from the ribosome causing a premature termination of polypeptide synthesis.
- Streptomycin causes misreading of the genetic code in bacteria at relatively low concentrations and inhibits initiation at higher concentrations, by binding to the 30s ribosomal subunit.
- Other aminoglycosides as Tobramycin and Kanamycin prevent ribosomal association at the end of initiation step and cause misreading of the genetic code.
- Tetracyclines block the A site on the ribosome, preventing the binding of aminoacyl tRNAs.
- Chloramphenicol blocks the peptidyl transfer step of elongation on the 50s ribosomal subunit in both bacteria and mitochondria.
- Macrolides and Lincosamides bind to the 50s ribosomal subunits inhibiting the peptidyltransferase reaction or translocation or both.
[edit] Eukaryotic translation
[edit] Initiation
[edit] The cap-dependent initiation
Initiation of translation involves an interaction of some proteins with a special tag bound to 5'-end of the mRNA molecules. The protein factors bind the small ribosomal subunit. The subunit accompanied by some of those protein factors moves along the mRNA chain towards its 3'-end and scans for the 'start' codon (mostly AUG) on the mRNA, which indicates where the mRNA starts coding for the protein. The sequence downstream between the 'start' and 'stop' codons is then translated by the ribosome into the aminoacid sequence -- thus a protein is synthesized. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine. The initiator tRNA charged with Met forms part of the ribosomal complex and thus all proteins start with this amino acid (unless it is cleaved away by a protease in some subsequent steps).
[edit] The cap-independent initiation
The best studied example of the cap-independent mode of translation initiation in eukaryotes is the Internal Ribosome Entry Site IRES approach. What differentiates cap-independent translation from cap-dependent translation is that cap-independent translation does not require the ribosome to start scanning from the 5' end of the mRNA cap until the start codon. The ribosome can be trafficked to the start site by ITAFs (IRES trans-acting factors) bypassing the need to scan from the 5' end of the untranslated region of the mRNA. This method of translation has been recently discovered, and has found to be important in conditions that require the translation of specific mRNAs, despite cellular stress or the inability to translate most mRNAs. Examples include factors responding to apoptosis, stress-induced responses.
cap-dependent/cap-independent initiation applies to both prokaryotes and eukaryotes.
[edit] Translation by hand
It is also possible to translate either by hand (for short sequences) or by computer (after first programming one appropriately, see section below), this allows biologists and chemists to draw out the chemical structure of the encoded protein on paper.
First, convert each DNA base to its RNA complement (note that the complement of A is now U):
DNA -> RNA A -> U T -> A G -> C C -> G
Then split the RNA into triplets (groups of three bases). Note that there are 3 translation "windows" depending on where you start reading the code. Finally, use the table at Genetic code to translate the above into a structural formula as used in chemistry.
This will give you the primary structure of the protein. However, proteins tend to fold, depending in part on hydrophilic and hydrophobic segments along the chain. Secondary structure can often still be guessed at, but the proper tertiary structure is often very hard to determine.
This approach may not give the correct amino acid composition of the protein, in particular if unconventional amino acids such as selenocysteine are incorporated into the protein, which is coded for by a conventional stop codon in combination with a downstream hairpin (SElenoCysteine Insertion Sequence, or SECIS).
[edit] Translation by computer
An enormous amount of computer programs capable of translating a DNA/RNA sequence into protein sequence exist. Normally this is performed using the Standard Genetic Code, and it's a safe bet that most Bioinformaticians have written at least one such program at some point in their education. However, few programs can handle all the "special" cases, such as the use of the alternative initiation codons: For examle the rare alternative start codon TTG codes for Methionine when used as a start codon and for Leucine in all other positions.
Example: Condensed translation table for the Standard Genetic Code (from the NCBI Taxonomy webpage).
AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = ---M---------------M---------------M---------------------------- Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
[edit] Translation tables
Even when working with ordinary Eukaryotic sequences such as the Yeast genome, it is often desired to be able to use alternative translation tables -- namely for translation of the mitochondrial genes. Currently the following translation tables are defined by the NCBI Taxonomy Group for the translation of the sequences in GenBank:
1: The Standard Code 2: The Vertebrate Mitochondrial Code 3: The Yeast Mitochondrial Code 4: The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code 5: The Invertebrate Mitochondrial Code 6: The Ciliate, Dasycladacean and Hexamita Nuclear Code 9: The Echinoderm and Flatworm Mitochondrial Code 10: The Euplotid Nuclear Code 11: The Bacterial and Plant Plastid Code 12: The Alternative Yeast Nuclear Code 13: The Ascidian Mitochondrial Code 14: The Alternative Flatworm Mitochondrial Code 15: Blepharisma Nuclear Code 16: Chlorophycean Mitochondrial Code 21: Trematode Mitochondrial Code 22: Scenedesmus obliquus mitochondrial Code 23: Thraustochytrium Mitochondrial Code
[edit] Software examples
- ApE (Mac, Windows, Unix)
- DNA Strider (Mac)
- ExPASy Translate Tool (webserver)
- Virtual Ribosome (webserver, cross-platform command-line)
Example of computational translation - notice the indication of (alternative) start-codons:
VIRTUAL RIBOSOME
----------------
Translation table: Standard SGC0
>Seq1
Reading frame: 1
M V L S A A D K G N V K A A W G K V G G H A A E Y G A E A L
5' ATGGTGCTGTCTGCCGCCGACAAGGGCAATGTCAAGGCCGCCTGGGGCAAGGTTGGCGGCCACGCTGCAGAGTATGGCGCAGAGGCCCTG 90
>>>...)))..............................................................................)))
E R M F L S F P T T K T Y F P H F D L S H G S A Q V K G H G
5' GAGAGGATGTTCCTGAGCTTCCCCACCACCAAGACCTACTTCCCCCACTTCGACCTGAGCCACGGCTCCGCGCAGGTCAAGGGCCACGGC 180
......>>>...))).......................................))).................................
A K V A A A L T K A V E H L D D L P G A L S E L S D L H A H
5' GCGAAGGTGGCCGCCGCGCTGACCAAAGCGGTGGAACACCTGGACGACCTGCCCGGTGCCCTGTCTGAACTGAGTGACCTGCACGCTCAC 270
..................)))..................)))......))).........)))......)))......))).........
K L R V D P V N F K L L S H S L L V T L A S H L P S D F T P
5' AAGCTGCGTGTGGACCCGGTCAACTTCAAGCTTCTGAGCCACTCCCTGCTGGTGACCCTGGCCTCCCACCTCCCCAGTGATTTCACCCCC 360
...)))...........................))).........))))))......)))..............................
A V H A S L D K F L A N V S T V L T S K Y R *
5' GCGGTCCACGCCTCCCTGGACAAGTTCTTGGCCAACGTGAGCACCGTGCTGACCTCCAAATACCGTTAA 429
...............))).........)))..................)))...............***
Annotation key:
>>> : START codon (strict)
))) : START codon (alternative)
*** : STOP
[edit] See also
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| Biochemical Processes | Molecular Biology Processes |
| Amino acid synthesis | tRNA synthesis | Transcription | Translation |
[edit] References
- ^ Structure fo the E. coli protein-coducting channel bound to at translating ribosome, K. Mitra, et al. Nature (2005), vol 438, p 318
- Pamela C Champe, Richard A Harvey and Denise R Ferrier (2005). Lippincott's Illustrated Reviews: Biochemistry (3rd ed.). Lippincott Williams & Wilkins. ISBN 0-7817-2265-9.
- David L. Nelson and Michael M. Cox (2005). Lehninger Principles of Biochemistry (4th ed.). W.H. Freeman. ISBN 0-7167-4339-6.
- Hirokawa et al. (2006) "The Ribosome Recycling Step: Consensus or Controversy?". Trends in Biochemical Sciences Vol. 31(3), 143-149.
