Download Analysis of the LacI family of repressor proteins in non

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.