Companies: Tevard Biosciences (TRNA therapies)

See Prokaryotics for differences with their translation

Translation is the process of converting the nucleotide sequence of mRNA into a protein. This is accomplished in the cell by using a genetic code consisting of codons which are sequences 3 nucleotides long. Each codon specifies 1 of the 20 different amino acids during protein synthesis. Since there are 4 possible nucleotides (A,C,G, U) there are 64 possible combinations of 3 nucleotides which make up a protein. These 64 possible combinations code for all of the 20 amino acids commonly used in protein synthesis which means that the code is degenerate in that more than 1 codon codes for the same amino acid. This degeneracy of the code also means that nucleotide mutations, particularly in the 3rd position of the codon often do not change the amino acid sequence. This is particularly true in transitions where one purine is replaced by another purine (i.e., A to G or G to A) or where one pyrimidine is replaced by another pyrimidine. However, transversions where a pyrimidine is substituted for a purine or vice versa typically is less protected in the genetic code and leads to altered amino acid sequences.

Definitions:

Initiation Codon: also referred to as the “AUG condon,” the “start condon” or the “AUG start codon” is typically 5’AUG (in transcribed mRNA molecules; 5’ATG in the corresponding DNA m olecule).

Translation termination codon also called the “stop codon” may have 1-3 sequences, i.e., 5’UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5’TAG and 5’TGA.

ORF: open reading frame also called “coding region” is known in the art to refer to the region between the translation initation codon and the translation termination codon. The first codon in an ORF is the “start” condon, which encodes a modified form of methionine. Each amino acid in the polypeptide chain is encoded by a subsequent set of three base pairs, until the translation is terminated at the stop codon that does not itself encode an amino acid, but rather signals the end of translation. Thus a double-tranded lenght of DNA can have six different reading frames, depending on the starting base-pair of the first codon and the direction in which the strand is read (two strands times three base pairs per codon equals six reading frames).

The codons in a mRNA molecule do not directly recognize the amino acids they specify. Instead, the translation of mRNA into protein depends on adaptor molecules called transfer RNAs (tRNAs) that can recognize and bind both to the codon at one end using 3 nucleotides called the anticodon and to the appropriate amino acid at the other end. The anticodon region of some tRNAs is sequenced such that they require accurate base pairing with the codon at the first two positions of the codon but can tolerate a mismatch at the 3rd position. For example, a special possible anticodon base, inosine (I) can recognize U, C or A. This “wobble” base-pairing between codons and anticodons accounts for degeneracy in the genetic code (the same amino acid can be specified by different codons). Inosine is just one of the many modified nucleotides on tRNA. TRNAs are transcribed by RNA polymerase III. 

Recognition and attachment of the correct amino acid to a tRNA depends on enzymes called aminoacyl-tRNA synthetases. For most cells there is a different synthetase enzyme for each amino acid. In this linkage process, the amino acid is first activated through the linkage of its carboxyl group to an AMP moiety using ATP. The AMP-linked carboxyl group on the amino acid is then transferred to an OH of the terminal A on the sugar at the 3′ end of the tRNA molecule using an activated ester linkage. 

Protein synthesis occurs by the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Thus a protein is synthesized from its N-terminal end to its C terminal end. To maintain the correct reading frame and to ensure accuracy, synthesis is carried out in the ribosome which is a complex consisting of more than 50 different proteins and several RNA molecules, the ribosomal RNAs. Eukaryotic and procaryotic ribosomes are very similar. Both are composed of one large and one small subunit that is divided up into further subunits. Ribosomal components are usually designated by their S values which refers to their rate of sedimentation in an ultracentrifuge.

A ribosome contains 4 binding sites for RNA molecules. One is for the mRNA and three called the E-site, P-site and A-site (“EPA) are for tRNAs. Initiation of translation occurs with the codon AUG and a special initiator tRNA which carries the amino acid, methionine (in bacterial, a modified form of methione–formylmethionine — is used). This Met will later typically be removed from the protein with a protease.

In eucaryotes, the initiator tRNA which is coupled to Met is loaded into the small ribosomal subunit along with additional proteins called eukaryotic initiation factors (eIFs). One such initiating factor, iIF-2, forms a complex with GTP and mediates the binding of the methionyl initiator tRNA to the small ribosomal subunit, which then binds to the 5′ end of the mRNA and begins scanning along the mRNA. When an AUG codon is recognized, the bound GTP is hydrolyzed to GDP by the eIF-2 protein, causing a conformation change in the eIF-2 protein and releasing it from the small ribosomal subunit. The large ribosomal subunit then joins the small one to form a complete ribosome that beings protein synthesis.

At this point, the initiator tRNA is bound to the P-site. The process of amino acid addition to form a protein occurs in a series of repeated steps. (1) a tRNA carrying the next amino acid in the chain binds to the ribosomal A-site by forming base pairs with the codon in mRNA which is positioned at the A-site. The aminoacyl-tRNA is tightly bound to and elongation factor (EF-Tu) which pairs transiently with the codon at the A site. The codon-anticodon pairing triggers GTP hydrolysis by EF-Tu causing it to dissociate from the tRNA. This delay between tRNA binding increases the accuracy of translation. At this point, the A and P sites contains adjacent tRNAs. (2) the carboxyl end of the polypeptide chain is released from the tRNA at the P-site and joined to the free amino group of the amino acid linked to the tRNA at the A site, forming a new peptide bond in a reaction catalyzed by peptidyl transferase. This reaction is accompanied by conformational changes in the ribosome which shift the tRNA into the E and P sites. (3) Additional conformational changes moves the mRNA exactly 3 nucleotides through the ribosome and resets the ribosome so it is ready to receive the next amino acyl tRNA.

The end of translation is signaled by one of 3 stop codons (UAA, UAG, or UGA) which are not recognized by a tRNA. Proteins called release factors (which mimic the shape and charge of a tRNA) bind to the ribosome with a stop codon positioned in the A site causing the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. This frees the carboxyl end of the growing polypeptide chain from its attachment to a tRNA.

In eukaryotes, translation can occur either in the cytoplasm or on the RER. Proteins that are translated on the RER are targeted there based on their own initial amino acid sequence. The ribosomes found on the RER are actively translating and are not permanently bound to the ER. A polypeptide that starts with a short series of amino acids called a signal sequence is specifically recognized and bound by a cytoplasmic complex of proteins called the signal recognition particle (SRP). The complex of signal sequence and SERP is in turn recognized by a receptor protein in the ER membrane.

Protein synthesis requires a lot of energy. At least 4 high energy phosphate bonds are split to make each new peptide bond. Two are consumed in charging a tRNA with an amino acid and two more drive steps in peptide synthesis. It would not only be a waste of energy therefore if incomplete mRNAs were translated but it would also be very harmful to the cell because aberrant proteins would be produced. Eukaryotes avoid this mistake by recognizing the 5′ cap and the poly A tail. Bacteria must solves this problem another way since there are no signals at the 3′ ends of bacterial mRNAs. Instead, the bacterial ribosome translates to the end of an incomplete RNA and then a special RNA, tmRNA, enters the A site of the robosome and is itself translated. This adds a special 11 amino acid to the C terminus of the truncated protein that signals for degradation.

One deviation from the genetic code is the use of a 21st amino acid called selenocysteine that can be incorporated into a growing polypeptide chain through translational recoding. Selenocysteine is essential for the efficient function of a variety of enzymes and is produced when a specialized tRNA is charged with serine which is then converted enzymatically. A specific RNA structure in the mRNA (a stem and loop structure with a particular nucleotide sequence) signals that selenocysteine is to be inserted at a UGA stop codon.

Another deviation from the genetic code that is produced by translational recording is called translational frameshifting which is commonly used by retroviruses like  HIV, in which it allows more than one protein to be synthesized from a single mRNA. These viruses commonly make both the capsid proteins (Gag proteins) and the viral reverse transcriptase and integrase (Pol proteins) from the same RNA transcript. Such a virus needs more copies of the Gag proteins and it achieves this by encoding the pol genes just after the gag genes but in a different reading frame. A stop codon at the end of the gag coding sequence can by bypassed on occasion. The frameshift occurs because features in the local RNA structure cause tRNAleuattached to the C terminus of the growing polypeptide chain to slip backward on occasion by one nucleotide on the ribosome.