Mutagenesis of an Arabinose Inducible Promoter - Lab Report Example

Biology Degree Second Year Genetics: Mutagenesis of an Arabinose Inducible Promoter To explore the regulatory functions of the arabinose operon in E.coli, a series of site-specific mutations was introduced into the PBAD promoter in a recombinant pGLO plasmid construct that included the AraC gene as well as the inducible promoter that regulates the expression of structural gene products required for the metabolism of arabinose. The mutant plasmids were introduced into E.coli by transformation and the effects on gene expression were measured by the production of fluorescence by the pGLO reporter gene. The results indicated that the mutations in the PBAD promoter DNA sequences affected the inducibility of gene expression by arabinose, either resulting in a loss of transcriptional capability or a switch to constitutive expression, based on the location of the inserted mutations. Introduction The promoter, the DNA binding site for RNA polymerase, regulates the initiation of messenger RNA synthesis. The DNA binding site consists of a core consensus sequences that are recognized by RNA polymerase as well as regulatory proteins that control promoter access by RNA polymerase. The arabinose inducible promoter controls the initiation of transcription of the structural genes responsible for the metabolism of the sugar arabinose which are synthesized only when the sugar substrate arabinose is present in the cell (Schlief, 2000). The arabinose operon (see Figure 1) includes both the structural genes and the regulatory genes that are involved in the regulation of expression from this group of genes. The structural genes are AraA (an isomerase), AraB (a ribulokinase) and AraD (an epimerase); each plays a role in the metabolism of arabinose to D-xylulose phosphate. The arabinose operon contains two promoters: PBAD and Pc. PBAD controls the transcription of the structural gene products of Ara-A, B and-D and Pc controls expression of AraC (Schlief, 2000). The AraC gene product functions as a repressor at the PBAD promoter, such that it is capable of dimerising at its N-terminal domains to generate a rigid N-terminal bridge or arm that binds DNA via its c-terminal domain to block transcription and translation of the structural gene products (Gryzcynski & Schlief, 2004). In the presence of arabinose, however, the rigid N-terminal arm binds to the inducer which causes a conformational change such that the repressor is no longer able to bind the PBAD promoter efficiently; thus, the site can be bound by RNA polymerase to initiate transcription of the structural gene products. The purpose of this experiment was to explore the effects of site-directed mutagenesis of the arabinose PBAD promoter on expression of the pGLO reporter gene due to altered binding of the AraC repressor in the mutant constructs. Figure 1. Organization and regulation of the arabinose-inducible promoter. Results Construction of mutant pGLO plasmids. Using the site-directed mutagenesis protocol, we constructed four mutant plasmids. The locations and sizes of the mutations are indicated in Figure 2. Figure 2. DNA sequence of site-directed mutagenesis products. M1-M4. 1. M2. CàT This has been reported to be a derepression mutation (Martin et al., 1986). 2. M4. GCàTT G and C represent AraC contacted guanines (Hendrickson, 1985) 3. M7. GàA This has been reported to be a derepression mutation (Martin et al., 1986) 4. M8. TàG Converts 19th codon of GFP from TTA to TGA (a STOP codon). Effects of mutations on pGLO expression in transformed E.coli. M8. Stop codon in GFP (Results from Chloe and Julliette.) Mutant colonies showed no fluorescence (relative to wild-type) in the presence of arabinose and displayed wild-type phenotype in the absence of arabinose. Figure 3. Results of M8 mutagenesis on pGLO fluorescent protein expression in E. coli transformants. M8 WT M8 WT (+) arabinose (-) arabinose M2. CàT in O2 (Results from Kelly and Matt.) Mutants show increased fluorescence (relative to wild-type) in absence of arabinose and unaltered fluorescence in presence of arabinose. Figure 4. Results of M2 mutagenesis on pGLO fluorescent protein expression in E.coli transformants. M2 WT (-) arabinose (-) arabinose (+) arabinose (+) arabinose M4. GCàTT in I1 Mutants showed less fluorescence in presence of arabinose and fluorescence in the absence of arabinose appeared unaltered. Figure 5. Results of M4 mutagenesis on pGLO fluorescent protein expression in E.coli transformants. (Results from SCFBC.) M4 WT WT M4 (+) arabinose (-) arabinose (Results from JS and RM.) WT M4 WT M4 (+) arabinose (-) arabinose M7. GàA in I2 (Results from Ryan and Rachel.) M7 mutants showed less fluorescence in presence of arabinose while fluorescence in the absence of arabinose appeared unaltered. Figure 6. Results of M7 mutagenesis on pGLO fluorescent protein expression in E.coli transformants. (+) arabinose m7 WT (-) arabinose M7 WT (-) ara (+) ara M7 WT (-)arabinose (+) arabinose M7 WT Discussion The study of gene regulation in bacteria serves as an important model for transcriptional regulation. In both the bacterial and eukaryotic systems, the control of transcription initiation represents an essential regulatory mode that determines the type and amount of proteins that are produced in a cell. The results of these experiments indicated that mutations in DNA sequences within identified regulatory and structural regions of the arabinose operon may have a dramatic effect on gene expression. This experiment utilised a recombinant pGLO plasmid constructed such that the expression of GFP was under the transcriptional control of the AraC regulatory protein and the arabinose-inducible PBAD promoter. A series of mutations was introduced into the recombinant pGLO plasmid by site-directed mutagenesis. One of these mutations, designated M8, was a point mutation that converted the 19th codon of GFP reporter gene from TTA to TGA (a STOP codon). The resulting nonsense mutation in the coding region of this gene prevented the expression of the fluorescent GFP protein in arabinose (+) cells despite the presence of an intact promoter. Nonsense mutations produce a premature termination of translation resulting in a failure to produce functional gene product. The presence of fluorescence was observed in the positive wild-type control in the presence of arabinose, but not when arabinose was absent from the culture medium, which indicated that the inducible promoter was functioning properly in this experimental system. M2 contained a point mutation that converted C (cytosine) to T (thymine) in the O2 binding site of the Ara2 repressor. The results indicated that the mutation caused a loss of inducible regulation of gene expression, as the synthesis of GFP occurred regardless of whether arabinose was present or not in the culture medium. In contrast, the wild-type promoter required arabinose for inducible expression. The most likely explanation for this observation was that the mutation altered the affinity of the DNA binding site for the AraC repressor protein, thereby permitting the constitutive expression of GFP regardless of the presence or absence of the inducer (Schlief, 2003). The change in DNA sequence resulted in a loss of regulated gene expression from the PBAD promoter. This result is consistent with previous research indicating that this represents a de-repression mutation (Ross, Gryczynski, & Schlief, 2003). The 02 regulatory site is located some distance from the PBAD promoter itself; however, when bound to the AraC dimer it generates a folded DNA loop structure that blocks RNA polymerase from binding to the promoter to initiate transcription (Schlief & Wolberger, 2004). The mutation in 02 may block Ara C interaction with this site so that the inhibitory loop does not form, resulting in an open promoter configuration accessible to RNA polymerase binding and transcriptional de-repression. The M4 mutation produced a dinucleotide substitution converting GC to TT. Previous research has determined that the G (guanine) and C (cytosine) dinucleotides represent part of a critical binding site for the AraC protein in a regulatory site of the PBAD promoter designated I1. The observed effect of the M2 mutation was a partial block to the inducible expression of GFP. This may result from a change in the promoter binding site that causes a less efficient binding of RNA polymerase to activate transcription even when the AraC repressor is bound by arabinose (Weldon & Schlief, 2006). The data suggested that the induction of GFP expression by arabinose was negatively affected by the mutation in the I1 regulatory site. The M7 mutation was characterised as a guanine (G) to adenine (A) point mutation in the I2 regulatory region. The effect of this mutation was a partial loss of inducible GFP production in the bacterial cells transformed with the mutant construct. This result was similar to that obtained with the mutation in the I1 binding site of the PBAD promoter which also resulted in a partial loss of inducible GFP expression. Other research studies have shown that the binding of the inducer arabinose to the AraC protein changes the affinity of AraC to the regulatory binding sites, such that the complex associates with I1 and I2 regulatory sites of the promoter in conjunction with the cyclic AMP (cAMP) binding cyclic AMP activator protein (CAP) to enhance the binding of RNA polymerase to the PBAD promoter to initiate transcription (Rogers, Larkin, & Schleif, 2007). The net result of I1 and I2 site interaction with the AraC–arabinose complex is to increase the binding of RNA polymerase to the PBAD promoter which enhances the rate of transcription of the adjacent structural genes. The mutations in I1 and I2 regulatory sites may block this interaction thereby resulting in a partial loss of inducibility of the PBAD promoter in the presence of arabinose. The net effect is a decrease in the production of gene product, evidenced by the lower level of fluorescence observed following lactose induction in these mutants. The data suggest that the I1 and I2 regulatory sites in the PBAD promoter may function in a manner similar to eukaryotic enhancer elements that regulate the level of transcription from associated promoters when bound to their regulatory protein activators (Timmes, Rodgers, & Schlief, 2004). The physical association of activated enhancers with their associated promoters strengthens the association of the promoter complex with RNA polymerase to achieve a higher level of transcriptional activity from adjacent genes (Schlief, 2004). The bacterial system may serve as a model for the complex promoter associated enhancer regulators that regulate the expression of eukaryotic genes in a tissue specific manner. In conclusion, the results of this experimental study demonstrated that site-directed mutagenesis using a reporter gene as an indicator of gene expression represents a powerful tool for exploring the regulatory functions of DNA sequences that interact with transcriptional activators and repressors via site-specific recognition binding domains. References Grycynski, U., and Schlief, R. (2004). A portable allosteric mechanism. Proteins 57, 9-11. Rogers, M., Larkin, C., & Schleif, R. (2007). Structure and properties of a truly Apo form of AraC dimerization domain. Proteins 66, 646-654. Ross, J., Gryczynski, U., & Schlief, R. (2003). Mutational analysis of residue roles in AraC function. Journal of Molecular Biology 328, 85-93. Schlief, R. (2000). Regulation of the L-arabinose operon of Escheria coli. Trends in Genetics 16, 559-565. Schlief, R. (2003). The AraC protein: a love-hate relationship. BioEssays 25, 274-282. Schlief, R. (2004). Building family traditions. Molecular Microbiology 53, 355-356. Schlief, R., & Wolberger, C. (2004). Arm-domain interactions can provide high binding cooperativity. Protein Science 13, 2829-2831. Timmes, A., Rodgers, M., & Schlief, R. (2004). Biochemical and physiological properties of the DNA binding domain of AraC protein. Journal of Molecular Biology 340, 731-738. Weldon, J., and Schlief, R. (2006). Specific interactions by the N-terminal arm inhibit self-association of the AraC dimerization domain. Protein Science 15, 2828-2835. Read More