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Regulation of Gene Expression. Information flow and regulatory factors. The number of copies of the gene The efficiency with which the gene is transcribed, which is mainly determined by the level of initiation of transcription by RNA polymerase (promoter activity). The stability of the mRNA
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The number of copies of the gene • The efficiency with which the gene is transcribed, which is mainly determined by the level of initiation of transcription by RNA polymerase (promoter activity). • The stability of the mRNA • The efficiency with which the mRNA is translated into protein • The stability of the protein product • Post-translational effects
Transcriptional control Promoters Most E. coli promoters, for example, have two key parts (motifs) that are involved in the recognition by the RNA polymerase and resemble TTGACA and TATAAT at positions that are centred at 35 bases and 10 bases before (upstream from) the transcriptional start site and are hence referred to as the -35 and -10 positions respectively. The latter is also known as the Pribnow box
Importance of the distance between the -35 and -10 regions of a promoter
Structure of the lac operon Operons and regulons
divergent genes In some cases, co- ordinated control of several genes is achieved by a single operator site that regulates two promoters facing in opposite directions In one example, the genes ilvC (coding for an enzyme needed for isoleucine and valine biosynthesis) and ilvY (which codes for a regulatory protein) are transcribed in opposite directions , but transcription of both genes is controlled by a single operator site This provides an exception to the general rule that genes on an operon are transcribed into a single mRNA Operons and regulons
Alternative promoters and σ-factors The promoter consensus described above is recognized by the primary s-factor, commonly referred to as σ70 (because it is about 70 kDa in size in E. coli). This subunit is responsible for recognition of the promoters used for transcription of most of the genes required in exponentially growing cells. These are sometimes called ‘housekeeping’ genes since they encode essential functions needed for the cell cycle and for normal metabolism such as glycolysis, the TCA cycle and DNA replication
Alternative sigma factors and promoter recognition sequences.In B.subtilis. All others are in E. coli
Sporulation in Bacillus At the onset of starvation, the primary σ-factor (σA) and a low abundance factor called σH direct the transcription of a set of genes whose products cause an asymmetric invagination of the membrane, thus separating the forespore from the mother cell Another σ-factor, σF, is present before the septum forms, but is inactive. σF becomes active but only in the forespore Following this, a third sporulation specific σ-factor, σE, also becomes active, but only in the mother cell
Anti-s-factors The regulation of flagella filament production (FliC) by the anti sigmafactor FlgM
Attenuation within an operon. The presence of a weak transcriptional termination site (t1) within an operon leads to reduced expression of the distal genes (c and d). The strong terminator t2 causes termination at the end of th full-length mRNA Terminators, attenuators and anti-terminators
The lac repressor is a multimeric protein, consisting of four identical subunits, showing a secondary structure feature consisting of two a-helices separated by a few amino acids that place the two a-helices at a defined angle to each other. This conformation, known as a helix-turn-helix motif, is characteristic of DNA-binding proteins and enables this part of the protein to fit into the major groove of the DNA and make specific contacts with the operator DNA
Regulation of the lac operon The lac repressor protein also has affinity for allolactose (a derivative of lactose) gratuitous inducer is the synthetic analogue iso-propyl-thiogalactoside (IPTG). The converse is also true: some compounds are substrates for breakdown by b- galactosidase but are not able to act as inducers since they are not recognized by the repressor. as X-gal (which gives a blue colour after hydrolysis by b- galactosidase)
Regulatory mutants of the lac operon Constitutive mutants (a) lacI mutants which are defective in the production of the repressor (or the repressor cannot bind to the operator) (capable of acting in trans) (b) operator-constitutive (Oᶜ) mutants in which the change is in the operator itself, preventing recognition by the repressor protein (These mutations are described as cis-dominant) Non-inducible mutants, which again are unaffected by the presence or absence of the inducer, but in this case the level of the enzymes is always low. The most significant one from our point of view is a different type of lacI mutation that abolishes the. ability of the repressor protein to recognize and respond to the inducer Super-repressor (lacIq) mutants. These cells are characterized by an overproduction of the repressor, commonly due to a mutation in the promoter of the lacI gene (remember that the lacI gene is not part of the lac operon and is transcribed from a different promoter).
Catabolite repression In the presence of glucose the level of ATP within the cell rises as the glucose is broken down to release energy; at the same time, the level of cyclic AMP (cAMP), a cellular alarm molecule, decreases due to activation of cAMPphosphodiesterase In the absence of glucose, adenylate cyclase is activated and levels of cAMP rise. Binding of cAMP to CRP causes a conformational change in the protein which allows it to recognize and bind to, specific sites on the DNA. The cAMP–CRP complex binds to a DNA site upstream from the promoter (-72 to -52) Despite the term catabolite repression, it should be clear that the role of the CRP is a positive one; it is an activator (when bound to cAMP) not a repressor. Hence, some people prefer to call it catabolite activator protein (CAP)
Protein binding sites in the regulatory region of the lac operon
Arabinose operon Repression and activation of the arabinose operon NH2-terminal domain binds arabinose and mediates dimerization, while the COOH-terminal domain contains the regions that bind to the DNA. When arabinose binds to the NH2-terminal domain, it alters the way that the dimer forms and hence its ability to contact different sites on the DNA. Thus in the absence of arabinose AraC is a negative regulator while in the presence of the substrate it acts as a positive regulator
Attenuation: trp operon Structure of the trp operon The operon contains a sequence (of 162 bases), known as the leader sequence, between the transcription start point and the start of the first structural gene
Attenuation control of the trp operon. (a) In the absence of protein synthesis, the terminator stem–loop 3:4 is able to form, and the operon is not transcribed. (b) If protein synthesis occurs in the presence of limiting amounts of tryptophan, ribosomes will stall at the tryptophan codons in the leader region, blocking formation of the 1:2 stem–loop. When the RNA polymerase transcribes region 3, it will pair with region 2. The 2:3 structure is not a terminator, but it sequesters region 3, thus preventing formation of the 3:4 terminator and allowing transcription of the operon. (c) In the presence of sufficient tryptophan, the ribosomes will proceed as far as the stop codon, thus blocking both regions 1 and 2. This allows the 3:4 termination structure to form, preventing transcription of the operon
Attenuation control of the trp operon in Bacillus subtilis. (a) The leader region contains two pairs of complementary sequences, enabling two alternative stem–loop structures A:B and C:D. The partial overlap of B and C prevents both structures forming. The leader also contains 11 repeats of GAG or UAG, which can bind TRAP (trp RNA-binding Attenuation Protein) in the presence of tryptophan. (b) In the absence of tryptophan, TRAP does not bind, the A:B stem–loop forms and the terminator C:D cannot form. Thus transcription of the operon occurs. (c) In the presence of tryptophan, TRAP binds to the GAG/UAG repeats, which blocks region A and prevents the formation of the A:B stem–loop. Region C is free to pair with region D to form the terminator structure, preventing transcription of the operon. Shaded circles indicate regions that are blocked from forming stem–loop structures
