No abstract

The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non‐cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non‐cognate DNA, stacked end‐to‐end, are bound to the dimeric enzyme in B‐DNA‐like conformations. The protein‐‐DNA interactions of this complex are prototypic for non‐specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B‐form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base‐specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co‐operative as they are all made through one short surface loop comprising residues 182–186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.

Cystals have been obtained on nucleosome cores and analysed by X-ray diffraction and electron microscopy. The core is a flat particle of dimensions about 110 X 110 X 57 A, somewhat wedge shaped, and strongly divided into two 'layers', consistent with the DNA being wound into about 1 3/4 turns of a flat superhelix of a pitch about 28 A. The organisation of the DNA can be correlated with the results to enzyme digestion studies. A change in the screw of the DNA double helix on nucleosome formation can be deduced.

Analysis of the ammo acid sequence of transcnptlon factor TFIIIA from Xenopus laevzs reveals the presence of 12 repeating structures, each about 30 residues m length These segments have been ahgned and their secondary structure predicted The repeats each contam two invariant cystemes and two invariant hlstldmes, perhaps to coordinate a zmc catlon Possible nucleic acid mteractlon modes are discussedTranscrrptlon factor TFIIIA

X-ray diffraction data from monoclinic crystals of yeast tRNAPhe soaked in dilute lead(II) acetate solutions at pH 5.0 and at pH 7.4 have been collected to a resolution of 3 A, and the Pb(II) binding sites have been obtained by difference Fourier analyses. The same three Pb(II) binding sites are observed at both of these pH values. At pH 7.4 an extra peak of negative electron density appears on the difference map close to one of the Pb(II) binding sites and at the position of phosphate-18, indicating cleavage of the sugar-phosphate-chain between residues D-17 and G-18 of the tRNAPhe molecule in this derivative. Chain scission does not occur to any observable extent in the structure at pH 5.0, and we have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. Polyacrylamide gel electrophoresis as well as sequencing experiments confirms the cleavage of the tRNAPhe molecule into one-fourth and three-fourth fragments, with the shorter fragment consisting essentially of residues G-1 through D-17 while the larger fragment contains residues G-18 through A-76. End-group analyses suggest a ribose cyclic 2',3'-phosphate at D-17 of the one-fourth fragment with a 5'-OH at G-18 of the three-fourth fragment. Cleavage of the tRNAPhe molecule does not occur in the absence of Pb(II), and the proximity of one of these metal ions to the cleavage site strongly implicates this metal ion in the cleavage reaction. Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2'-OH of ribose-17 by a Pb(II)-bound hydroxyl group. Subsequent nucleophilic attack of the resulting 2'-O- on the phosphorus atom of phosphate-18, presumably through a pentacoordinate phosphorus cyclic intermediate (as in the action of pancreatic ribonuclease A), results in chain scission. It cannot be decided whether the displacement, within the pentacoordinate intermediate, proceeds via an in-line or adjacent pathway, but an exploration of the likelihood of either pathway is presented. Strand cleavage at the particular site occurs fortuitously because the aquo Pb(II) ion binds at the correct distance and presumably in such a manner as to present a hydroxyl group in the correct orientation to effect the proton abstraction.(ABSTRACT TRUNCATED AT 400 WORDS)