Translation refers to the process of polymerisation of amino acids to form a polypeptide. The order and sequence of amino acids are defined by the sequence of bases in the mRNA. The amino acids are joined by a bond which is known as a peptide bond. Formation of a peptide bond requires energy. Therefore, in the first phase itself amino acids are activated in the presence of ATP and linked to their cognate TRNA-a process commonly called as charging of tRNA or aminoacylation of tRNA to be more specific. If two such charged tRNAs are brought close enough, the formation of peptide bond between them would be favored energetically. The presence of a catalyst would enhance the rate peptide bond formation The cellular factory responsible for synthesising proteins is the ribosome. The ribosome consists of structural RNAS and about 80 different proteins. its inactive state, it exists as two subunits; a large subunit and a small subunit.
When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. There are two sites in the large subunit, for subsequent amino acids to bind to and thus, be close enough to each other for the fornmation of a peptide bond. The ribosome also acts as a catalyst (23S rRNA in bacteria is the enzyme- ribozyme) for the formation of peptide bond. A translational unit in mRNA is the sequence of RNA that is flanked by the start codon (AUG) and the stop codon and codes fora polypeptide. An mRNA also has some additional sequences that are not translated and are referred as untranslated regions (UTR). The UTRS are present at both 5-end (before start codon) and at 3′-end (after stop codon). They are required for efficient translation process. For initiation, the ribosome binds to the mRNA at the start codon (AUG) that is recognised only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into Polypeptide sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.
Regulation of gene expression
Regulation of gene expression refers to a very broad term that may occur At various levels. Considering that gene expression results in the formation of a polypeptide, it can be regulated at several levels. In eukaryotes, the regulation could be exerted at
- Transcriptional level (formation of primary transcript).
- Processing level (regulation of splicing).
- transport of MRNA from nucleus to the cytoplasm,
- Translational level.
The genes in a cell are expressed to perform a particular function or a set of functions. For example, if an enzyme called beta-galactosidase is synthesised by E. coli, it is used to catalyse the hydrolysis of a disaccharide, lactose into galactose and glucose; the bacteria use them as a source of energy. Hence, if the bacteria do not have lactose around them to be utilised for energy source, they would no longer require the synthesis of the enzyme beta-galactosidase. Therefore, in simple terms it is the metabolic, physiological or environmental conditions that regulate the expression of genes. The development and differentiation of embryo into adult organisms are also a result of the coordinated regulation of expression of several sets of genes. In prokaryotes, control of the rate of transcriptional initiation is the predominant site for control of gene expression. In a transcription unit, the activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulatory proteins can act both positively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator bind a repressor protein. Each operon has its specific operator and specific repressor. For example, lac operator is present only in the lac operon and it interacts specifically with lac repressor only.
The Lac operon
The elucidation of the lac operon was also a result of a close association between a geneticist, Francois Jacob and a biochemist, Jacque Monod. They were the first to elucidate a transcriptionally regulated system. In lacoperon (here lac refers to lactose), a polycistronic structural gene is regulated by a common promoter and regulatory genes. Such arrangement is very common in bacteria and is referred to as operon. To name few such examples, lac operon, trp operon, ara operon, his operon, val operon, etc. The lac operon consists of one regulatory gene (the i gene – here the term i does not refer to inducer, rather it is derived from the word inhibitor) and three structural genes (z, y, and a). The igene codes for the repressor of the lac operon. The z gene codes for beta-galactosidase (B-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to B-galactosides. The a gene encodes a transacetylase. Hence, all the three gene products 11 lac operon are required for metabolism of lactose. In most other operons as well, the genes present in the operon are needed together to function in the same or related metabolic pathway
The lac Operon Lactose is the substrate for the enzyme beta-galactosidase ana it In the absence of a preferred carbon source such as glucose, if lactose is provided ii the growth medium of the bacteria, the lactose is transported into the cells through the action of permease (Remember, a very low level of expression of lac operon has to be present in the cell all the time, otherwise lactose cannot enter the cells). The lactose then induces the operon in the following manner. The repressor of the operon İs synthesised (all-the-time- constitutively) from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. In the presence of an inducer, such as lactose or allolactose, the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the promoter and transcription proceeds. Essentially, regulation of lac operon can also be visualised as regulation of enzyme synthesis by its substrate. Remember, glucose or galactose cannot act as inducers for lac operon. Can you think for how long the lac operon would be expressed in the presence of lactose? Regulation of lac operon by repressor is referred to as negative regulation. Lac operon is under control of positive regulation as well, but it is beyond the scope of discussion at this level.