MoBC-translation.pdf

An mRNA Sequence Is Decoded in Sets of Three Nucleotides The nucleotide sequence of a gene, through the intermediary of mRNA, is translated into the amino acid sequence of a protein by rules that are known as the genetic code. This code was deciphered in the early 1960s. Since there are only 4 diffe...

An mRNA Sequence Is Decoded in Sets of Three Nucleotides The nucleotide sequence of a gene, through the intermediary of mRNA, is translated into the amino acid sequence of a protein by rules that are known as the genetic code. This code was deciphered in the early 1960s. Since there are only 4 different nucleotides in mRNA and 20 different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein. The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three = codons there are 4 × 4 × 4 = 64 possible combinations of three nucleotides. However, only 20 different amino acids are commonly found in proteins. Either some nucleotide triplets are never used, or the code is redundant and some amino acids are specified by more than one triplet. The second possibility is, in fact, the correct one.. In principle, an RNA sequence can be translated in any one of three different reading frames, depending on where the decoding process begins. These are called Open Reading Frames (ORF) However, only one of the three possible reading frames in an mRNA encodes the required protein tRNA Molecules Match Amino Acids to Codons in mRNA The translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), each about 80 nucleotides in length. Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically Two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis. One of these regions forms the anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. The other is a short single-stranded region at the 3ʹ end of the molecule; this is the site where the amino acid that matches the codon is attached to the tRNA. Aminoacyl-tRNA Synthetases Couple Each Amino Acid to Its Appropriate tRNA Molecule Aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. Most cells have a different synthetase enzyme for each amino acid (that is, 20 synthetases in all); one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. The wobble position While there are 64 different types of codons there are not 64 different types of t-RNA. This is because some t-RNA can recognize more than one codon. This usually occurs because there is non-canonical base pairings (like G:U) in the third position of the codon and the first position of the anticodon (wobble position). If inosine (a modified adenine that can pair with A, G and U) is on the wobble position then that t-RNA can recognize A, G and U in the third position of the codon) Aminoacyl-tRNA Synthetases links the C-terminus of an amino acid to the 3’end of the t-RNA Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain Ribosomes will catalyze the peptide bond between the N-terminus of an amino acid bound to a t-RNA and the C-terminus of the growing polypetide chain that bound to the previous t-RNA This is the reason why the first amino acid in proteins are the N-terminus The RNA Message Is Decoded in Ribosomes The ribosome is a complex catalytic machine made from more than 50 different proteins (the ribosomal proteins) and several RNA molecules, the ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm The large and small ribosome subunits are assembled at the nucleolus, where newly transcribed and modified rRNAs associate with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm. These two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins. A ribosome contains four binding sites for RNA molecules A ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A site, the P site, and the E site) are for tRNAs A ribosome contains four binding sites for RNA molecules A ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A site, the P site, and the E site) are for tRNAs A tRNA molecule is held tightly at the A and P sites only if its anticodon forms base pairs with a complementary codon (allowing for wobble) on the mRNA molecule that is threaded through the ribosome The A and P sites are close enough together for their two tRNA molecules to be forced to form base pairs with adjacent codons on the mRNA molecule. This feature of the ribosome maintains the correct reading frame on the mRNA. Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing four major steps: tRNA binding (step 1): tRNA carrying the next amino acid in the chain binds to the ribosomal A site by forming base pairs with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. peptide bond formation (step 2): the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by breakage of the high-energy bond between the tRNA and its amino acid) and joined to the free amino group of the amino acid linked to the tRNA at the A site, forming a new peptide bond. This central reaction of protein synthesis is catalyzed by a peptidyl transferase contained in the large ribosomal subunit large subunit translocation (step 3): the large subunit moves relative to the mRNA held by the small subunit, thereby shifting the acceptor stems of the E and P sites of the large subunit small subunit translocation (step 4): another series of conformational changes moves the small subunit and its bound mRNA exactly three nucleotides, ejecting the spent tRNA from the E site and resetting the ribosome so it is ready to receive the next aminoacyl-tRNA Elongation Factors Drive Translation Forward and Improve Its Accuracy Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process. These factors are called EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes. Without the aid of these elongation factors and GTP hydrolysis, translation is very slow, inefficient, and inaccurate. Coupling the GTP hydrolysis-driven changes in the elongation factors to transitions between different states of the ribosome speeds up protein synthesis enormously. These factors: -Provide energy necessary during translation -Help bringing the right t-RNA that match the corresponding anti-codon -Help remove t-RNA wrongly associated with the corresponding anti-codon (proofreading) -Makes the process go forward and never backwards Sequences in mRNA Signal Where to Start Protein Synthesis The site at which protein synthesis begins on the mRNA is especially crucial, since it sets the reading frame for the whole length of the message. An error of one nucleotide either way at this stage would cause every subsequent codon in the message to be misread, resulting in a nonfunctional protein with a garbled sequence of amino acids. The translation of an mRNA begins with the codon AUG, and a special tRNA is required to start translation. This initiator tRNA always carries the amino acid methionine, with the result that all newly made proteins have methionine as the first amino acid at their N-terminus, the end of a protein that is synthesized first. The initiator tRNA is specially recognized by initiation factors because it has a nucleotide sequence distinct from that of the tRNA that normally carries methionine Sequences in mRNA Signal Where to Start Protein Synthesis In eukaryotes, the initiator tRNA–methionine complex (Met–tRNAi) is first loaded into the small ribosomal subunit along with additional proteins called eukaryotic initiation factors, or eIFs. Of all the aminoacyl-tRNAs in the cell, only the methionine-charged initiator tRNA is capable of tightly binding the small ribosome subunit without the complete ribosome being present, and unlike other tRNAs it binds directly to the P site. Next, the small ribosomal sub- unit binds to the 5ʹ end of an mRNA molecule, which is recognized by virtue of its 5ʹ cap that has previously bound two initiation factors, eIF4E and eIF4G The small ribosomal subunit then moves forward (5ʹ to 3ʹ) along the mRNA, searching for the first AUG.. Sequences in mRNA Signal Where to Start Protein Synthesis At this point, the initiation factors dissociate, allowing the large ribosomal subunit to assemble with the complex and complete the ribosome. The initiator tRNA remains at the P site, leaving the A site vacant. Protein synthesis is therefore ready to begin The nucleotides immediately surrounding the start site in eukaryotic mRNAs influence the efficiency of AUG recognition during the above scanning process. If this recognition site differs substantially from the consensus recognition sequence (5ʹ-ACCAUGG-3ʹ), scanning ribosomal subunits will sometimes ignore the first AUG codon in the mRNA and skip to the second or third AUG Stop Codons Mark the End of Translation The end of the protein-coding message is signaled by the presence of one of three stop codons (UAA, UAG, or UGA). These are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. Proteins known as release factors bind to any ribosome with a stop codon positioned in the A site, forcing the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. This reaction frees the carboxyl end of the growing polypeptide chain from its attachment to a tRNA molecule, and since only this attachment normally holds the growing polypeptide to the ribosome, the completed protein chain is immediately released into the cytoplasm. The ribosome then releases its bound mRNA molecule and separates into the large and small subunits. These subunits can then assemble on this or another mRNA molecule to begin a new round of protein synthesis. Proteins Are Made on Polyribosomes The synthesis of most protein molecules takes between 20 seconds and several minutes. During this very short period, however, it is usual for multiple initiations to take place on each mRNA molecule being translated. As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, the 5ʹ end of the mRNA is threaded into a new ribosome. The mRNA molecules being translated are therefore usually found in the form of polyribosomes (or polysomes): large cytoplasmic assemblies made up of several ribosomes spaced as close as 80 nucleotides apart along a single mRNA molecule. These multiple initiations allow the cell to make many more protein molecules in a given time than would be possible if each protein had to be completed before the next could start. In eukaryotes, the 5ʹ and 3ʹ ends of the mRNA interac; therefore, as soon as a ribosome dissociates, its two subunits are in an optimal position to reinitiate translation on the same mRNA molecule Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics. Some of these drugs exploit the structural and functional differences between bacterial and eukaryotic ribosomes so as to interfere preferentially with the function of bacterial ribosomes. Thus, humans can take high dosages of some of these compounds without undue toxicity There Are Many Steps From DNA to Protein