Abstract

To understand how proteins recognize specific sequences on double-stranded DNA, and to understand how this binding affects gene function, we propose to undertake an X-ray crystallographic study of the lambda repressor-operator complex and a crystallographic study of the arc repressor from phage p22. The co-crystals that we propose to study contain the N-terminal domain of lambda repressor and a 20 base pair duplex that includes the intact lambda operator site. These crystals diffract to at least 3.0 A resolution. The crystals of the arc protein (which is interesting because it does not show a significant sequence homology with the three DNA-binding proteins of known structure) diffract to 2.2 A resolution. In addition to solving these two structures, we propose to: 1) grow co-crystals that contain the intact lambda repressor and the lambda cro protein, 2) grow co-crystals that contain the arc repressor, 3) grow co-crystals that contain lambds repressor and/or operator mutations, and 4) begin crystallographic study of eukaryotic regulatory proteins. The first eukaryotic protein that we will attempt to crystallize is the C-terminal domain of the Epstein-Barr virus nuclear antigen. This domain binds specifically to the origin of DNA replications.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031471-04
Application #
3279476
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1983-01-01
Project End
1990-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Grant, R A; Rould, M A; Klemm, J D et al. (2000) Exploring the role of glutamine 50 in the homeodomain-DNA interface: crystal structure of engrailed (Gln50 --> ala) complex at 2.0 A. Biochemistry 39:8187-92
Pabo, C O; Nekludova, L (2000) Geometric analysis and comparison of protein-DNA interfaces: why is there no simple code for recognition? J Mol Biol 301:597-624
Xu, H E; Rould, M A; Xu, W et al. (1999) Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding. Genes Dev 13:1263-75
Chasman, D; Cepek, K; Sharp, P A et al. (1999) Crystal structure of an OCA-B peptide bound to an Oct-1 POU domain/octamer DNA complex: specific recognition of a protein-DNA interface. Genes Dev 13:2650-7
Fraenkel, E; Rould, M A; Chambers, K A et al. (1998) Engrailed homeodomain-DNA complex at 2.2 A resolution: a detailed view of the interface and comparison with other engrailed structures. J Mol Biol 284:351-61
Pomerantz, J L; Wolfe, S A; Pabo, C O (1998) Structure-based design of a dimeric zinc finger protein. Biochemistry 37:965-70
Tucker-Kellogg, L; Rould, M A; Chambers, K A et al. (1997) Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity. Structure 5:1047-54
Klemm, J D; Pabo, C O (1996) Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. Genes Dev 10:27-36
Xu, W; Rould, M A; Jun, S et al. (1995) Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations. Cell 80:639-50
Ma, P C; Rould, M A; Weintraub, H et al. (1994) Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell 77:451-9

Showing the most recent 10 out of 33 publications