Translation The basic processes of translation are conserved among prokaryotes and eukaryotes. Prokaryotic Translation A ribosome, mrna, and trna. In the initiation of translation in prokaryotes, the Shine-Dalgarno sequence binds to a complementary sequence in 16S rrna. The initiation complex forms following the movement of the small subunit to the first AUG codon, which sets the reading frame. In prokaryotes, the methionine of initiator trna is modified to block the amino end with a formyl group. When the 50S subunit associates with an initiation complex, the initiator trna is located in the P-site. During the elongation phase of translation, charged trnas enter through the A-site, peptide bonds are formed between the amino acid at the P-site and the amino acid at the A-side, 10
trna deacetylase breaks the bond between a trna and its amino acid in the P-site, and uncharged trnas leave the ribosome at the E-site. The decoding centre of a ribosome. The peptidyl transferase centre of a ribosome and the action of trna deacetylase. Translation stops when a stop codon enters the A-site and a release factor enters the A-site. When translation stops, IF3 dissociates the ribosomal subunits. In prokaryotes, translation can occur at the same time as transcription. Eukaryotic Translation Translation initiation in eukaryotes is dependent on a 5 cap binding to eif4e, which is a capbinding protein. When eif4e binds to a 5 cap, it recruits a number of proteins, including eif4a, which is a helicase. 11
A number of proteins prevent the 40S subunit from binding to the 60S subunit when translation is not occurring. In eukaryotes, initiator trna associates with a 40S subunit before the 40S subunit associates with mrna to form the 43S pre-initiation complex. In eukaryotes, the methionine of initiator trna is not modified to block the amino end with a formyl group. The 48S pre-initiation complex forms when the 43S pre-initiation complex binds to mrna and its associated eif proteins. In eukaryotes, the start codon must be in the context of a Kozak sequence for the 60S subunit to associate with the 40S subunit. Cap-binding proteins associate with poly(a)-binding proteins, and so eukaryotic mrna is translated as a circular molecule, which aids the repeated translation of an mrna molecule. In eukaryotes, transcription and translation are separated in time and space. Operons In eukaryotes, translation initiation usually depends on a 5 cap, so most eukaryotic mrna is monocistronic. In prokaryotes, many genes are arranged as operons, which are expressed on a single polycistronic mrna molecule. 12
The lac operon. Nonsense Mutations A mutation that produces an early stop codon results in a nonsense mutation, which are usually loss-of-function mutations. The effect of a nonsense mutation can be suppressed by a mutation in a trna anticodon. Polar lacz mutants have a nonsense mutation in their lacz gene that prevents the translation of the lacy gene and the laca gene by causing the dissociation of ribosomes, which allows Rho to terminate transcription. In eukaryotes, nonsense mediated decay (NMD) removes transcripts in which a nonsense mutation has stopped translation prematurely. Nonstop Mutations If a stop mutation has been mutated, ribosomes will proceed until they reach a stop codon or the end of the mrna. If a ribosome reaches the end of a molecule of mrna, it stalls. In E. coli, tmrna enters stalled ribosomes, functions as trna and as mrna, tags the polypeptide for degradation with a specific amino acid sequence, and allows the ribosome to dissociate from the mrna and the polypeptide. 13
Effect of Mutation on Protein Structure The frequency and dominance of particular mutations can provide information about the function of a gene. The conservation of an amino acid sequence at a region of a protein suggests that that region is important in the function of the protein. Amino Acids Aspartic acid (Asp) and glutamic acid (Glu) have acidic side chains. Lysine (Lys), arginine (Arg), and histidine (His) have basic side chains. Asparagine (Asn) and glutamine (Gln) have side chains containing an amide group. Serine (Ser) and threonine (Thr) have side chains containing a hydroxyl group. Phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) have aromatic side chains. Alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), and glycine (Gly) have nonpolar side chains. Cysteine (Cys) and methionine (Met) have side chains containing sulphur. Hierarchy of Protein Structure The primary structure of a protein is the linear sequence of its amino acids. The secondary structure of a protein consists of alpha helices, beta sheets, turns, loop structures, and supersecondary structures. The tertiary structure of a protein consists of domains, which are regions of a polypeptide chain that can fold independently of the rest of the polypeptide chain. The quaternary structure of a protein consists of multiple polypeptide chains. Mutations in Noncoding Regions A mutation in a promoter or a regulatory region may affect the synthesis of a gene. A mutation in an intron may affect protein structure if the mutation alters the splicing of the intron. Rarely, a mutation in an intergenic region may affect gene expression. 14
Mutations in Coding Regions A mutation in a coding region may be a silent mutation, a nonsense mutation, a missense mutation, an addition mutation, or a deletion mutation. Silent mutations, some missense mutations that alter a non-critical region of the resulting protein, and some conservative missense mutations have no detectable effect on the function of the resulting protein. Frameshift mutations, nonsense mutations, and most missense mutations will cause a lossof-function phenotype. Loss-of-function mutations are usually recessive. Some mutations, such as mutations that result in temperature sensitive mutants, may have a conditional phenotypical effect. Mutations with conditional effects are usually conservative missense mutations. Gain-of function mutations are usually missense mutations, are usually dominant, and are not common. Dominant negative mutations are usually in genes that code for the monomer of a homooligomer. 15
Suppression Suppression occurs when the effect of a mutation is phenotypically counteracted by another mutation. A revertant phenotype may be the result of a reversion, the result of intragenic suppression, or the result of extragenic suppression. Intragenic suppression can be the result of a frameshift suppression or missense suppression. Extragenic suppression Translational suppression occurs when a nonsense mutation is suppressed by a mutation in the anticodon of a trna that causes the trna to bind to a stop codon. Translational suppression is not totally efficient, since mutated trna will be in competition with release factors. An example of physiological suppression. An example of physiological suppression. 16
An example of protein-protein interaction suppression. Protein-protein interaction suppression usually involves missense mutations. An example of gene expression suppression. Characterising Revertants If a revertant phenotype is the result of a reversion, a cross between a revertant and a wild type will not produce progeny with a mutant phenotype. If a revertant phenotype is the result of intragenic suppression, a cross between a revertant and a wild type will rarely produce progeny with a mutant phenotype. If a revertant phenotype is the result of extragenic suppression, a cross between a revertant and a wild type will readily produce progeny with a mutant phenotype. 17