Gene expression of prokaryotes is controlled basically at two levels i.e. transcription and translation stages. In addition, mRNA degradation and protein modification also play a role in regulation. Most of the prokaryotic genes that are regulated are controlled at transcriptional stage. Other control measures operating at different levels are given in Table.2.4.
It is a general strategy in a living organism that chemical changes occur by a metabolic pathway through a chain of reactions. Each step is determined by the enzyme.
Again synthesis of an enzyme comes under the control of genetic material i.e. DNA in living organisms. Enzyme (protein) synthesis occurs via two steps : transcription and translation. Transcription refers to synthesis of mRNA. Transcription is regulated at or around promoter region of a gene. By controlling the ability of RNA polymerase to the promoter the cell can modulate the amount of message being transcribed through the structural gene. However, if RNA polymerase has bound, it again can modulate transcription. By doing so the amount of gene product synthesized is also modulated. The coding region is also called structural gene. Adjacted to it are regulatory regions that control the structural gene. The regulatory regions are composed of promoter (for the initiation of transcription) and an operator (where a diffusible regulatory protein binds) regions.
The molecular mechanisms for each of regulatory patterns vary widely but usually fall in one of two major groups: negative regulation and positive regulation. In negative regulation an inhibitor is present in the cell and prevents transcription. This inhibitor is called as repressor. An inducer i.e. antagonist repressor is required to permit the initiation of transcription. In a positive regulated system an effector molecule (i.e. a protein, molecule or molecular complex) activates a promoter. The repressor proteins produce negative control, whereas the activator proteins produce positive control. Since the transcription process is accomplished in three steps polymerase binding, isomerization of a few nucleotides and release of RNA polymerase from promoter region), the negative regulators usually block the binding, whereas the activators interact with RNA polymerase making one or more steps. In negative regulation an inhibitor is bound to the DNA molecule. It must be removed for efficient transcription. In positive regulation an effector molecule must bind to DNA for transcription.
Table 2.4. Types of gene regulation in prokaryotes operating at different levels.
The Lac Operon
For the first time Jacob and Monod (1961) gave the concept of operon mode! to explain the regulation of gene action. An operon is defined as several distinct genes situated in tadem all controlled by a common regulatory region) Commonly an operon consists of repressor, promoter, operator and structural genes. The message produced by an operon is polycistronic because the information of all the structural genes resides on a single molecule of mRNA.
The regulatory mechanism of operon responsible for utilization of lactose as a carbon source is called the lac operon.lt was extensively studied for the first time by Jacob and Monod (1961). Lactose is a disaccharide which is composed of glucose and galactose.
The lactose utilizing system consists of two types of components; the structural genes (lacZ, LacY and lac A) the products of which are required for transport and metabolism of lactose and the regulatory genes (the lacI, the lacO and the lacP). These two components together comprises the lac operon (Fig. 2.11). One of the most key features is that operon provides a mechanism for the coordinate expression of structural genes controlled by regulatory genes. Secondly, operon shows polarity i.e. the genes Z,Y and A synthesize equal quantities of three enzymes b-galactosidase (by lacZ), permease ( by lacY) and acetylase (by lacA). These are synthesized in an order i.e. b-galactosidase first and acetylase in the last.
(i) The structural genes. The structural genes form one long polycistronic mRNA molecule. The number of structural gene corresponds to the number of proteins. Each structural gene is controlled independently, and transcribe mRNA molecules separately. This depends on substrates to be utilized. For example, in lac operon three structural genes (Z,Y and A) are associated with lactose utilization (Fig. 2.11). b-galactose is the product of lacZ that cleaves b-1,4 linkage of lactose and releases the free monosaccharides. This enzyme is a tetramer of four identical subunits each with molecular weight of 1,16,400. The enzyme permease (a product of lacY) facilitates the lactose to enter inside the bacterium. Permease has molecular weight of 46,500. It is hydrophobia The cells mutant in lacZ and lacY are designated as Lac" i.e. the bacteria cannot grow in lactose. The enzyme transacetylase (30,000 MW) is a product of lac A whose no definite role has been assigned.
This section of two fold symmetry is present on the CRP site that binds to a protein called CRP (cyclic AMP receptor protein). The CRF is encoded by CRP gene. It has been shown experimentally that CRP binds to cAMP (cyclic AMP found in E. coli and other organisms) molecule and form a cAMP-CRP complex. This complex is required for transcription because it binds to promoter and enhances the attachment of RN A polymerase to the promoter. Therefore, it increases transcription and translation processes. Thus, cAMP-CRP is a positive regulator in contrast with the repressor, and the lac operon is controlled both positively and negatively.
According to a model proposed by Pribnow (1975) the promoter region consists of three important components which are present at a fixed position to each other. These components are (i) the recognition sequence, (ii) the binding sequence, and (iii) an mRNA initiation site. The recognition sequence is situated outside the polymerase binding site that is why it is protected from DNase. Firstly, RNA polymerase binds to DNA and forms a complex with the recognition sequence. The binding site is 7 bp long (5'TATGTTG) and present at such region that is protected from DNase. In other organisms the base pairs do not differ from more than two bases. Hence, it can be written as 5' TATPuATG. The mRNA initiation site is present near the binding site on one of the two bases. The initiation site is also protected from DNase. However, there is overlapping of promoter and operator in lac operon. Moreover, there is a sequence 5'CCGG, 20 bp left to mRNA initiation site. This is known as Hpall site (5'CCGG) because of being cleaved at this site by the restriction enzyme HpaII.
(iv) The repressor (regulator) gene. Repressor gene determines the transcription of structural gene. It is of two types: active and inactive repressors. It codes for amino acid of a defined repressor protein. After synthesis the repressor molecules are diffused from the ribosome and bind to the operator in the absence of an inducer. Finally the path of RNA polymerase is blocked and mRNA is not transcribed. Consequently no protein synthesis occurs. This type of mechanism occurs in the inducible system of active repressor.
Moreover, when an inducer (e.g. lactose) is present it binds to repressor proteins and forms an inducer-repressor complex. This complex cannot bind to the operator. Due to formation of complex the repressor undergoes changes in conformation of* shape and becomes inactive. Consequently the structural genes can synthesize the polycistronic mRNAs and the later synthesizes enzymes (proteins). In contrast, in the reversible system the regulator gene synthesizes repressor protein that is inactive and, therefore fails to bind to operator. Consequently, proteins are synthesized by the structural genes. However, the repressor proteins can be activated in the presence of a corespressor. The corepressor together with repressor proteins forms the repressor-corepressor complex. This complex binds to operator gene and blocks protein synthesis.
Jacob and Monod (1961) could not identify the repressor protein. Gilbert and Muller - Hill (1966) succeeded in isolating the lac repressor from the lac mutant cells of E. coli inside which the lac repressor was about ten times greater than the normal cells. The lac repressor proteins have been crystallized. It has a molecular weight of about 1,50,000 daltons. It consists of four subunits each has 347 amino acid residues and molecular weight of about 40,000 daltons. The repressor proteins have strong affinity for a segment of 12-15 base pairs of operator gene. This binding of repressor blocks the synthesis of mRNA transcript by RNA polymerase.
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