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Studies on the active site of E. coli RNA polymerase
METALS
& GENE EXPRESSION
343
MO18
-IIGN CHIMETRY GF MEDULG-DERIVATIVES GF PI-IAGE T4 GENE 32 PROTEIN. D.P. Giedroc C. Robertson, & R. Khan, Texas A&M University, College Station, TX 77843-2128, USA. Gene 32 protein (g32P) binds single-stranded (ss) DNA substrates formed during T4 DNA replication and repair. G32P contains an intrinsic Zn” ion ligate# in a tetrahedral ligand field by the side chains CysT7, His” (proposed), Cys and Cyspo [l]. We have prepared both the Co” (d sf) and Ni” (d8) substituted g32Ps and have examined their functional activities and coordination structures Ni”, in contrast to Co”, through absorption, CD, MCD and NMR spectroscopies. clearly does not confer structural stability on the DNA binding domain and is defective in cooperative ssDNA binding. While the Co” is bound tetrahedrally, the Ni” appears bound in some alternate geometry; preliminary MCD and ‘H NMR data implicate a square planar (S=O) arrangement of ligands which would suggest a significant degree of structural elasticity in the metal binding loop region. [1] D.P. Giedroc et al., Biochemistry 28, 2410 (1989).
M()w
STRUCTURE AND FUNCTION OF THE ZN(II) BINDING SITE WITHIN THE DNA BINDING DOMAIN OF THE GAL4 TRANSCRIPTION FACTOR. T. Pan & Joseph .E. Coleman, Yale University, New Haven, CT 06510, USA. GAL4 from S. cerevisiae contains a Cys6 MZn-finger”-like motif within its DNA binding domain (l-74). A homogeneous GAL4 fragment (l- 147+2: GAL4( 149*)) overexpressed in E. coli contains 1 to 1Sgm atom of Zn(II)/molecule. GAL4( 149*) binds specifically to a 17bp consensus UASG sequence. Removal of the intrinsic Zn(I1) abolishes binding to this specific DNA; addition of Zn(II), Cd(I1) or Co(I1) to apo GAL4( 149*) restores specific DNA binding. 1 13Cd-NMR reveals two Cd(I1) binding sites on the molecule with 6 of 707ppm and 669ppm, both suggesting coordina-tion to 3 to 4 sulfur atoms. GAL4(149*) contains 40% a-helix and 20% B-sheet estimated from circular dichroism. Removal of the native Zn(II) ion results in unfolding of less than one turn of a-helix. The binding of Zn(II), Cd(I1) to apo GAL4(149*) protects the protein from proteolysis by trypsin which produces a 13kD DNA binding core.
M(-)20
STUDIES ON THE ACTIVE SITE OF E. COLI RNA POLYMERASE. Gunther L. Eichhorn, Peter Chuknyisky, Richard Beal, Joseph Rifkind, Raj Pillai, and Edward Tarien, NIH, NIA, Gerontology Res. Baltimore, Maryland 21224, USA. Ctr., We are determining the geometry of interaction by which the enzyme brings together at the active site DNA template and the substrates between which an internucleotide bond is formed in RNA synthesis. Each of the two substrates is located on a separate subsite and each subsite is bound to a metal ion, [Zn(JI) and Mg(II)]. After displacement of both metals by Mn(II), separately or simultaneously, distances between metals are determined by EPR, and between metals and points on the substrates by NMR techniques. We believe that the enzyme places the substrates in an optimal configuration for bond formation between substrates that correctly copy the DNA genetic code.
& GENE EXPRESSION
343
MO18
-IIGN CHIMETRY GF MEDULG-DERIVATIVES GF PI-IAGE T4 GENE 32 PROTEIN. D.P. Giedroc C. Robertson, & R. Khan, Texas A&M University, College Station, TX 77843-2128, USA. Gene 32 protein (g32P) binds single-stranded (ss) DNA substrates formed during T4 DNA replication and repair. G32P contains an intrinsic Zn” ion ligate# in a tetrahedral ligand field by the side chains CysT7, His” (proposed), Cys and Cyspo [l]. We have prepared both the Co” (d sf) and Ni” (d8) substituted g32Ps and have examined their functional activities and coordination structures Ni”, in contrast to Co”, through absorption, CD, MCD and NMR spectroscopies. clearly does not confer structural stability on the DNA binding domain and is defective in cooperative ssDNA binding. While the Co” is bound tetrahedrally, the Ni” appears bound in some alternate geometry; preliminary MCD and ‘H NMR data implicate a square planar (S=O) arrangement of ligands which would suggest a significant degree of structural elasticity in the metal binding loop region. [1] D.P. Giedroc et al., Biochemistry 28, 2410 (1989).
M()w
STRUCTURE AND FUNCTION OF THE ZN(II) BINDING SITE WITHIN THE DNA BINDING DOMAIN OF THE GAL4 TRANSCRIPTION FACTOR. T. Pan & Joseph .E. Coleman, Yale University, New Haven, CT 06510, USA. GAL4 from S. cerevisiae contains a Cys6 MZn-finger”-like motif within its DNA binding domain (l-74). A homogeneous GAL4 fragment (l- 147+2: GAL4( 149*)) overexpressed in E. coli contains 1 to 1Sgm atom of Zn(II)/molecule. GAL4( 149*) binds specifically to a 17bp consensus UASG sequence. Removal of the intrinsic Zn(I1) abolishes binding to this specific DNA; addition of Zn(II), Cd(I1) or Co(I1) to apo GAL4( 149*) restores specific DNA binding. 1 13Cd-NMR reveals two Cd(I1) binding sites on the molecule with 6 of 707ppm and 669ppm, both suggesting coordina-tion to 3 to 4 sulfur atoms. GAL4(149*) contains 40% a-helix and 20% B-sheet estimated from circular dichroism. Removal of the native Zn(II) ion results in unfolding of less than one turn of a-helix. The binding of Zn(II), Cd(I1) to apo GAL4(149*) protects the protein from proteolysis by trypsin which produces a 13kD DNA binding core.
M(-)20
STUDIES ON THE ACTIVE SITE OF E. COLI RNA POLYMERASE. Gunther L. Eichhorn, Peter Chuknyisky, Richard Beal, Joseph Rifkind, Raj Pillai, and Edward Tarien, NIH, NIA, Gerontology Res. Baltimore, Maryland 21224, USA. Ctr., We are determining the geometry of interaction by which the enzyme brings together at the active site DNA template and the substrates between which an internucleotide bond is formed in RNA synthesis. Each of the two substrates is located on a separate subsite and each subsite is bound to a metal ion, [Zn(JI) and Mg(II)]. After displacement of both metals by Mn(II), separately or simultaneously, distances between metals are determined by EPR, and between metals and points on the substrates by NMR techniques. We believe that the enzyme places the substrates in an optimal configuration for bond formation between substrates that correctly copy the DNA genetic code.