The objective of our research is to understand how regulatory proteins interact with DNA and therefore control gene expression. Our studies focus on two systems-the lac operon and the major rightward control region of bacteriophage lambda. Lac operators containing sequence and functional group modifications are being synthesized and biochemically analyzed. Similarly a large number of base analogs and sequence changes will be introduced into lambda PR. These substitutions will permit us to precisely map those functional groups on promoters that are recognized by E. coli RNA polymerase, lac, cI and cro repressors, and catabolite activator protein. The research so far suggests that lac repressor may not recognize a unique lac operator sequence. This research should also help define the steps involved in initiation of transcription, the mechanism of catabolite activation, and how two proteins (cro and cI) with quite different amino acid sequences recognize the same DNA sequence. Research will also be directed toward understanding the mechanism whereby catabolite activator protein stimulates transcription. Initially deletions and insertions will be introduced synthetically into the lac promoter at selected sites and the effect of these modifications will be measured in appropriate E. coli strains. Other research will focus on the amino acid residues in lac repressor and cro repressor that recognize specific DNA sequences. Lac repressor can be specifically cross linked with lac operator using bifunctional reagents. The crosslinking sites between DNA bases and amino acid side-chanis will be investigated. Cro repressor will be probed by site-specific mutagenesis. Amino acid changes will be introduced into cro by modifying the cro gene. The effect of these modifications on cro binding will be monitored in vivo and in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021120-13
Application #
3270262
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-12-01
Project End
1988-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
13
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Cummins, L; Graff, D; Beaton, G et al. (1996) Biochemical and physicochemical properties of phosphorodithioate DNA. Biochemistry 35:8734-41
Tonkinson, J L; Guvakova, M; Khaled, Z et al. (1994) Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: a comparative study. Antisense Res Dev 4:269-78
Wiesler, W T; Marshall, W S; Caruthers, M H (1993) Synthesis and purification of phosphorodithioate DNA. Methods Mol Biol 20:191-206
Marshall, W S; Caruthers, M H (1993) Phosphorodithioate DNA as a potential therapeutic drug. Science 259:1564-70
Caruthers, M H; Beaton, G; Wu, J V et al. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs. Methods Enzymol 211:3-20
Marshall, W S; Beaton, G; Stein, C A et al. (1992) Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine. Proc Natl Acad Sci U S A 89:6265-9
Caruthers, M H; Beaton, G; Cummins, L et al. (1991) Synthesis and biological studies with dithioate DNA. Nucleic Acids Symp Ser :91-4
Delannoy, P; Caruthers, M H (1991) Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract. Mol Cell Biol 11:3425-31
Hubbard, A J; Bracco, L P; Eisenbeis, S J et al. (1990) Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding. Biochemistry 29:9241-9
Caruthers, M H; Brill, W K; Grandas, A et al. (1989) Synthesis of oligodeoxynucleoside phosphorodithioates. Nucleic Acids Symp Ser :119-20

Showing the most recent 10 out of 17 publications