Sherlin LD, Bullock TL, Nissan TA, Perona JJ, LaRiviere F, Uhlenbeck OC & Scaringe S. Chemical and enzymatic synthesis of tRNAs forhigh-throughput crystallization. RNA, 7, 1671-1678 (2001). Abstract

Horton NC, Dorner LF & Perona JJ. Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. Nature Structural Biology 9, 42-47 (2002). Abstract

Newberry KJ, Hou Y-M & Perona JJ. Structural origins of amino acid selection without editing by cysteinyl-tRNA synthetase. EMBO J. 21, 2778-2787 (2002).

Horton NC, Otey C, Lusetti S, Sam MD, Kohn J, Martin AM, Ananthnarayan V & Perona JJ. Electrostatic contributions to site-specific DNA cleavage by EcoRV endonuclease. Biochemistry 41, 19754-19763 (2002).

Francklyn C, Perona JJ, Puetz J & Hou Y-M. Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation. RNA, 8, 1363-1372 (2002).

Perona JJ. Type II restriction endonucleases. Methods: A companion to Methods in Enzymology, 28, 353-364 (2002).

Zhang C-M, Christian T, Newberry K, Perona JJ & Hou YM. Zinc-mediated amino acid discrimination in cysteinyl-tRNA synthetase. J. Mol. Biol., 327, 911-917 (2003).

Bullock TL, Uter N, Nissan TA & Perona JJ. Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants. J. Mol. Biol., 328, 395-408 (2003).

Sherlin LD & Perona JJ. tRNA-dependent active-site assembly in a class I aminoacyl-tRNA synthetase. Structure, 11, 591-603 (2003).

Perona JJ. Glutaminyl-tRNA synthetase. In: The Aminoacyl-tRNA Synthetases (M. Ibba, C. Francklyn, S. Cusack, eds), Landes Bioscience, in the press (2003).

Hou YM & Perona JJ. Cysteinyl-tRNA synthetase. In: The Aminoacyl-tRNA Synthetases (M. Ibba, C. Francklyn, S. Cusack, eds), Landes Bioscience, in the press (2003).

Zhang C-M, Perona JJ & Hou YM. Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase. Biochemistry, in the press (2003).


(1) Sam MD, Horton NC, Nissan TA & Perona JJ. Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site. J. Mol. Biol., 306, 851-861 (2001). Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn2+ binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence. In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71. The new metal binding site is specific to Mn2+ and is not occupied in ternary cocrystal structures with either Mg2+ or Ca2+. Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn2+ sites significantly modulate activity toward both cognate and noncognate DNA substrates. Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn2+-dependent cleavage rates of specific substrates by ten-fold. Conversely, the enhancement of noncognate cleavage at GTTATC sequences by Mn2+ is significantly attenuated in the mutants. As a consequence, under Mn2+ conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC noncognate site. These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 � distant from the scissile phosphodiester linkages. They also show that discrimination of cognate versus noncognate DNA sequences by EcoRV must depend in part on contacts with the sugar-phosphate backbone outside of the target site.

(2) Sherlin LD, Bullock TL, Nissan TA, Perona JJ, LaRiviere F, Uhlenbeck OC & Scaringe S. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA, 7, 1671-1678 (2001). Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by Xray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 � resolution. Multimilligram quantities of tRNAs with greatly reduced 3'-heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically-modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 � resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA-synthetase complex permit the detailed structural study of "atomic-group" mutants.

(3) Horton NC, Dorner LF & Perona JJ. Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. Nature Structural Biology 9, 42-47 (2002). The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPy/PuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side-chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center Py-Pu step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base stacking free-energies are a crucial underlying factor mediating protein recognition by indirect readout.