Proteins produced from such DNA templates are called recombinant proteins. Therefore, DNA templates of specific genes, with or without add-on reporter or affinity tag sequences, can be constructed as templates for protein expression. Unlike proteins, DNA is simple to construct synthetically or in vitro using well established recombinant DNA techniques. Instead, living cells and their cellular machinery are usually harnessed as factories to build and construct proteins based on supplied genetic templates. Given the size and complexity of proteins, chemical synthesis is not a viable option for this endeavor. To investigate how particular proteins regulate biology, researchers usually require a means of producing (manufacturing) functional proteins of interest. In general, proteomics research involves investigating any aspect of a protein such as structure, function, modifications, localization or protein interactions. The initiation step is the rate-limiting step in eukaryotic translation. More than three initiation factors, which are regulated by phosphorylation. Three initiation factors are known, IF1, IF2, &IF3 Sometimes Kozak sequence may be present around the initiation codon.Ĭap-independent translation: Ribosome binding to mRNA occurs through 'internal ribosome entry site' (IRES) on mRNA. The first AUG codon in the 5’end of mRNA functions as the initiation codon. Translation initiation occurs in two ways:Ĭap-dependent translation: Cap structure and the cap binding proteins are responsible for proper ribosome binding to mRNA and recognition of the correct initiation codon. The first amino acid of the nascent polypeptide is formylated methionine. This facilitates binding and alignment of the ribosome on the mRNA at the translation initiation site (AUG). The Shine-Dalgarno sequence is present on the mRNA transcript, and a complementary sequence is present in the ribosomal subunit. The Cap structure and the poly A are important for export of mRNA to the cytoplasm, proper initiation of translation and stability of mRNA among other functions. MRNA is polycistronic and contains multiple initiation sites.Īfter transcription, the mRNA transcript is spliced to remove the noncoding regions (introns), and a cap structure (M7methyl guanosine) and a poly adenosine sequence are added at the 5' and 3' end of the message respectively. No further processing of mRNA transcript occurs after transcription. At this point, the ribosome is released from the mRNA and is ready to initiate another round of translation. Finally, the nascent polypeptide is released in the termination step when the ribosome reaches the termination codon. The sequence of amino acids added to the growing peptide is dependent on the mRNA sequence of the transcript. During elongation, tRNAs bind to their designated amino acids (known as tRNA charging) and shuttle them to the ribosome where they are polymerized to form a peptide. Protein factors as well as sequences in mRNA are involved in the recognition of the initiation codon and formation of the initiation complex. The large subunit of the ribosome joins the small ribosomal subunit to generate the initiation complex at the initiation codon. The overall process is similar in both prokaryotes and eukaryotes, although particular differences exist.ĭuring initiation, the small subunit of the ribosome bound to initiator t-RNA scans the mRNA starting at the 5’end to identify and bind the initiation codon (AUG). There are specific protein factors for each step of translation (see table below). Translation or protein synthesis is a multi-step process that requires macromolecules like ribosomes, transfer RNAs (tRNA), mRNA and protein factors as well as small molecules like amino acids, ATP, GTP and other cofactors. The modified mRNA is then exported to the cytoplasm where it is translated. In eukaryotes, however, mRNA is further processed to remove introns (splicing), addition of a cap at the 5´ end and multiple adenines at the mRNA 3´ end to generate a polyA tail. In prokaryotes, no special modification of mRNA is required and translation of the message starts even before the transcription is complete. Transcription is regulated at various levels by activators and repressors and also by chromatin structure in eukaryotes. Once transcription is initiated, RNA polymerase is released from the DNA. Transcription begins when the double-stranded DNA is unwound to allow the binding of RNA polymerase. Transcription occurs in three steps in both prokaryotes and eukaryotes: initiation, elongation and termination.
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