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Analysis of the LacI family of repressor proteins in nonlactose-utilizing bacteria Ann Matthysse, Edwin Wong, and L (Sandy) Pierson 2007 ASM/BioQuest Bioinformatics Institute Introduction: Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of 'structural' genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell (Lewis, 2005). Binding of the lacO operator region by the LacI repressor protein in E. coli is well studied. Blast analysis of soil borne Agrobacterium spp. and Pseudomonas spp. indicates that they also contain DNA binding repressors with sequence similarity to E. coli LacI. These soil borne bacteria do not utilize lactose as a carbon source. Therefore, these LacI family proteins are involved in negative regulation of alternative pathways. Approach: The protein structure of the N-terminal 62 amino acid residues of LacI bound to the lacO1 operator sequence has been solved at 2.6 Angstrom resolution. Analysis of the Nterminal end of LacI indicates that several amino acid residues are conserved to various degrees (Fig. 1). Figure 1. Three-dimensional representation of the Nterminal LacI fragment bound to the lacO1 operator site. Table 1: Conserved residues of the N-terminal region of LacI. Based on observations made in the PDB database. Highly Conserved A10 V15 S16 T19 S21 P49 N50 A53 Medium Conserved V30 R22 N25 Y47 L6 D8 T5 L56 Figure 2. DNA sequence of the lacO operator indicating regions of dyad symmetry (Lodish et al, 2000). The amino acid sequence of E. coli (-proteobacteria) LacI was used to Blast the Agrobacterium (-bacterium) and Pseudomonas (-proteobacteria) genomic web sites for similar proteins. A total of 13 ‘hits’ (7 Pseudomonas and 5 Agrobacterium) were downloaded into the Biology Workbench 3.2. The selected sequences were compared using the ClustalW phylogenetic program (Fig. 2). The ClustalW output was saved as a .PILEMSF file. Both rooted and unrooted trees were constructed using the default parameters (Fig. 4A & 4B). Analysis of the phylogenetic trees indicated that these proteins are not particularly closely related. However, closely related protein sequences from similar bacteria grouped most closely, as expected. A file of the aligned amino acid sequences was assembled (took waaaay toooooo looooong!), imported into ConSurf and compared to the LacI PDB file. Comparison of the output in which the amino acid residues of LacI were compared to the other proteins indicated that 7 residues were highly conserved, 3 were moderately conserved, and 10 were somewhat conserved, while the rest showed little sequence similarity. The most highly conserved residues were clustered in groups surrounding one or the other lacO1 DNA strand (A10, G14, S16, S21, A41, P49, A53). The moderately conserved residues (T19, N50, L56) appear to join the highly conserved residues and the somewhat conserved residues (V15, D8, K37, N25, S31, R22, Q54, M42, Y47, R35) were generally located away from the DNA strands and may join the N-terminal end to the rest of the repressor protein. The Hypothesis: Alterations in specific amino acid residues will have profound impacts on repressor-DNA binding affinity. Sub-hypothesis 1: Test: Using a lacPlacO::GFP reporter plasmid, measure the relative level of repression by each LacI homolog. Using site-specific mutagenesis, change amino acid residues predicted to be involved in DNA base recognition or structural aspects and measure the impact on GFP repression. Alternatively, error-prone PCR could be utilized to phenotypically screen for altered protein sequences that result in changes in the baseline regulation of the GFP reporter. As a subsequent experiment, a sacB cartridge could be introduced downstream of GFP. sacB encodes for a levan sucrase that converts sucrose into a toxic compound. Counter-selection against expression of sacB on 10% sucrose medium would select for second site mutations that increase repressor-operator binding affinity by the various repressors. The mutations could occur either in the gene encoding the repressor or in the operator sequence itself. Concluding Remarks: We hope that a development of this project would serve as the basis of several undergraduate honors research projects. This work would integrate hands-on bench experiments with bioinformatic analysis of structural aspects of DNAprotein interactions and repressor function. Subsequent experiments could be aimed at swapping regions of the repressors and seeing if responses to various effector molecules (sugars, etc) were predictably altered. Figure 3. Texshade comparison of LacI homologs in Pseudomonas and Agrobacterium spp. with E. coli LacI. Figure 4A. Unrooted phylogenetic tree of LacI homologs. Figure 4B. Rooted phylogenetic tree of LacI homologs. Figure 5. Comparison of the 13 selected amino acid sequences against E. coli LacI N-terminal protein using ConSurf with an imported MSA file. Residues colored dark purple are highly conserved across all the proteins examined while light purple and cyan colored residues are moderately conserved and less conserved, respectively. White residues are not conserved while yellow colored residues lack sufficient data to make a determination. DNA LacI Nterminal peptide Ala53 Ala10 Gly14 Ala13 Pro49 Ser21 Ser16 Leu56 Asn50 Thr19 Acknowledgements: To Sam and John who made all this fun and to Amy and Kelly for cookies. References: Bell, CE, and Lewis, M. 2000. A closer view of the conformation of the Lac repressor bound to operator. Nature Structural Biology 7, 209 - 214 doi:10.1038/73317 Lewis, M. 2005. The Lac repressor. C R Biol. 328:521-48. Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. In Molecular Cell Biology. 4th ed., New York: W. H. Freeman & Co.; c2000. Wilson CJ, Zhan H, Swint-Kruse L, Matthews KS. 2007. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding. Cell Mol Life Sci. 64:3-16. http://molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_anim_lar ge.jif.