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T A Steitz

Publications and source records attributed to T A Steitz.

At least 127 records · Page 7Linked to original sources
PubMed1986

Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli.

Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained. Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis. Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A. They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution. Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes.

Escherichia coli↗
PubMed1986

Crystallization of recA protein from Proteus mirabilis.

The recA protein from Proteus mirabilis, which is homologous to the Escherichia coli protein, forms crystals in the orthorhombic space group P2(1)2(1)2(1). There are two 38,000 molecular weight subunits in the asymmetric unit and the unit cell dimensions are a = 57.5, b = 127.0 and c = 157.0 A.

Crystallography↗
PubMed1986

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Binding Sites↗
PubMed1985

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

DNA Polymerase I↗
PubMed1985

Crystal structure of muconate lactonizing enzyme at 6.5 A resolution.

We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme. They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis. The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4. The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry.

Crystallography↗
PubMed1984

A model for the non-specific binding of catabolite gene activator protein to DNA.

The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA.

Base Composition↗
PubMed1984

Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity.

Calculation of the electrostatic potential energy surfaces of Escherichia coli catabolite gene activator protein (CAP) dimer suggests a model for the complex between CAP and a specific DNA sequence. The positive electrostatic charge density of CAP lies on the two COOH-terminal domains and about 20-30 A from the molecular 2-fold axis. Assuming that the 2-fold axes of the CAP dimer and the DNA to which it binds are coincident, the positions of the positive electrostatic potential surfaces strongly suggest the rotational orientation of the DNA relative to the protein. A specific complex between CAP and its DNA binding site in the lac operon has been built with the DNA in this orientation. The amino ends of the two protruding F alpha-helices interact in successive major grooves of the DNA. Four side chains emanating from each F helix can form hydrogen bonds with the exposed edges of four bases in the major groove. Electrostatic considerations as well as the necessity to make interactions between CAP and a DNA site as much as 20 base pairs long require us to bend or kink the DNA. In our model of CAP complexed with B-DNA, as with those proposed for Cro and lambda cI repressors, the protruding second helices of the two-helix motif from both subunits interact in successive major grooves of B-DNA. However, unlike Cro and similar to lambda cI, the protruding alpha-helices are nearly parallel to the bases rather than the groove.

Amino Acid Sequence↗
PubMed1984

Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically.

The 20,500-dalton gamma delta resolvase monomer can be cleaved by chymotrypsin into a 5000-dalton COOH-terminal fragment and a 15,500-dalton NH2-terminal fragment that have been purified. Two crystal forms of the large fragment have been obtained, one of which is isomorphous with crystals of the native protein, showing that the large fragment makes the protein-protein contacts in the crystal and that the small fragment is segmentally disordered relative to the large fragment. Nuclease protection demonstrates that the small fragment binds specifically to all three DNA binding sites protected by resolvase. However, unlike native resolvase, which binds to all three complete sites with equal affinity, the small fragment binds to each of the six half sites with a different affinity. It has not been possible to demonstrate specific DNA binding of the larger fragment. Thus, resolvase has a modular construction analogous to that found for some repressors and activators; its COOH-terminal domain recognizes specific sequences in the DNA and its NH2-terminal domain mediates protein-protein interactions and probably has the enzymatic activity.

Amino Acid Sequence↗
PubMed1983

Crystallization of a ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA and its complex with protein L25.

A ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA has been crystallized. The space group is P6(1)22 or P6(5)22, with a = 59.5 A and C = 268 A. The crystals contain one molecule per asymmetric unit, and show diffraction to 4.0 A resolution. Also, a complex of this fragment with L25 ribosomal protein has been crystallized in the same space group, but with a = 119 A, c = 250 A and four molecules per asymmetric unit.

Crystallization↗
PubMed1983

Crystallization and 7 A resolution electron density map of the large fragment of Escherichia coli DNA polymerase I.

Crystals of the large fragment of Escherichia coli DNA polymerase I have been grown that diffract to better than 2.8 A resolution. They are in tetragonal space group P4(3) with a = b = 104.1 A, c = 86 A. A 7 A resolution map shows the protein to consist of two domains and to be mostly alpha-helical. The active site has been located by binding nucleoside monophosphates.

Binding Sites↗
PubMed1983

Ligand-induced conformational changes in proteins.

The natures and roles of ligand-induced conformational changes are described in terms of the influence of the ligand on the equilibrium distribution of the protein structure among various conformational states. A very commonly observed conformational change is produced by a ligand binding into a cleft that lies between two domains and then closing the cleft through a hinge-like motion. Such a change in hexokinase serves to bring potential catalytic groups into the active site and to provide a binding site for the three phosphates of ATP. Flexibility in gene regulatory proteins such as the Escherichia coli catabolite activator protein and lac repressor may be important in the dual requirement for rapid diffusion of the proteins along the DNA as well as specific recognition of base sequences. A second role for flexibility in this class of proteins is to reduce their affinity for affinity for DNA, without reducing their capacity to discriminate among sequences.

Adenosine Triphosphate↗
PubMed1983

Crystallographic studies of protein-nucleic acid interaction: catabolite gene activator protein and the large fragment of DNA polymerase I.

Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor 'Core', 5S RNA fragment and its complex with L25. Calculation of the electrostatic charge potential of CAP, using coordinates refined at 2.6 A resolution, suggests an orientation for B DNA on this repressor and activator of transcription. Both the electrostatic calculations and detailed model building suggests that the DNA must be bent or kinked on the protein in this orientation in order to make sufficient protein contacts. From a 3.5 A resolution map of Pol I fragment we have been able to obtain a preliminary trace through the polypeptide backbone. The large fragment consists of two domains. The smaller domain binds nucleoside monophosphate at the edge of a mostly parallel beta-pleated sheet, a structure that is reminiscent of kinase and dehydrogenase nucleotide binding domains. The larger domain contains about two thirds of the fragment and is mostly alpha-helical but with at least one four stranded antiparallel beta-sheet. The nucleoside monophosphate binds with its 5' phosphate on the Mg and is apparently in the conformation of nucleotides in B DNA.

Cyclic AMP Receptor Protein↗
PubMed1982

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.

Amino Acid Sequence↗