- Email: [email protected]
The tertiary structure of tRNA and the development of the genetic code
TIBS 1 8 - O C T O B E R 1 9 9 3
of indefinitely extended polypeptide chains rather than discrete subunits linked partly by end-to-end interactions. In separate discussions of myosin and tropomyosin, no mention is made of any similarity to keratins. It would seem to be time for textbooks to present a unified discussion of the coiled coil as a distinct and widely occurring protein structure and to describe accurately the organization of proteins, including keratins, in which coiled coils occur.
References 1 Fuchs, E. et at. (1987) Curt. Top. Oev. Biol. 22, 5-34 2 Crick, F. C. (1953) Acta Cryst. 6, 689-697 3 Fraser,R. D. B. and MacRae,T. P. (1973) Conformation in Ifibrous Proteins, AcademicPress 4 Parry, D. A. D. (1990) in Cellular and Molecular Biology of Intermediate Filaments (Goldman,
The elucidation of the three-dimensional tRNA structure as an 'L' shape that folds from a cloverleaf secondary structure has an interesting outcome ~ (Fig. 1). The anticodon triplet and the amino acid attachment site are located at the opposite ends of the L, separated by about 75 A. In living cells, the connection that determines the relationship between amino acid attachment and the anticodon triplet is established by the 20 aminoacyl tRNA synthetases, one for each amino acid. This connection underlies the principles of the genetic code, where each of the 64 codon triplets that corresponds to a particular amino acid or stop instruction during protein synthesis is interpreted by tRNAs through nucleic acid base-pairing to the anticodon triplets. We have gained new insights into the question of how aminoacyl tRNA synthetases recognize tRNAs, in part because many synthetases have been found to specifically aminoacylate RNA oligonucleotides that derive from the acceptor stem sequences of the cognate tRNAs~. Because the acceptor stem is proximal to the amino acid attachment site, recognition by the synthetase can probably be accommodated by the same protein domain as interacts with the amino acid. However, a fraction of these synthetases also recognize the anticodon triplet for aminoacylation. The distal separation between the anticodon and the amino acid attachment site implies that the synthetases that
362
R. 0. and Steinert, P. M., eds), pp. 175-204, Plenum Press 5 Steinert, P. M. and Roop, D. R. (1988) Annu. Rev. Biochem. 57,593-625 6 Stewart, M. (1993) C~rr. Opin. Cell Biol, 5, 3-11 7 Cohen, C. and Parry, D. A. D. (1986) Trends Biochem. Sci. 11, 245-248 80'Shea, E. K., Klemm, J. D., Kim, P. S. and AIber, T. (1991) Science 254, 539-544 9 Conway, J. F. and Parry, D. A. D. (1991) Int. 1 Biol. Macremol, 13, 14-16 10 Lovejoy, 8. et al. (1993) Science 259, 1288-1293 11 Hatzfeld, M. and Weber, K. (1990) J. Ceil Biol. 110, 1199-1210 12 Steinert, P. M. (1990) J. Biol. Chem. 265, 8766-8774 13 Coulombe, P. A. and Fuchs, E. (1990) J. Cell Biol. 111, 153-169 14 Steinert, P. M. et al. (1991) Int. J. Biol. MacromoL 13, 130-139 15 Fraser, R. D. B., MacRae, T. P. and Parry, D. A. D. (1990)in Cellular and Molecular Biology of intermediate Filaments (Goldman, R. D. and Steinert, P. M., eds), pp, 205-231, Plenum Press
The tertiary structure of tRNA and the development of genetic code
16 Aebi, U., Fowler, W. E., Rew, P. and Sun, T. (1983) J. Ceil Biol. 97, 1131-1143 17 Engel, A., Eichner, E. and Aebi, U. (1985) J. Ultrastruct. Res. 90, 323-335 18 Steinert, P. M. (1991) J. Struct. Biol. 107, 157-174 19 Geisler, H., SchOnemann,J. and Weber, K. (1992) Eur. J. Biochem. 206, 841--852 20 Steven, A. C. (1990) in Cellular and Molecular Biology of Intermediate Filaments (Goldman, R. D. and Steinert, P. M., eds), pp. 233-263, Plenum Press 21 Lehninger, A. L., Nelson, D. L. and Cox, M. M. (1993) Principles of Biochemistry (2rid edn), Worth 22 Zubay, G. (1993) Biochemistry (3rd edn), Wm C. Brown 23 Matthews, C. K. and Van Holde, K. E, (1990) Biochemistry, Benjamin/Cummings
JEFFREY A. COHLBERG Department of Chemistry and Biochemistry, California State University, 1250 Bellflower Bird, Long Beach, CA 90840-0115, USA.
nucleotides that are in contact with the synthetase significantly decrease the catalytic efficiency of aminoacylation, and transfer of these nucleotides to a different tRNA sequence framework confers recognition of the latter by the synthetase 7-9, The ability to change tRNA identity by switching defined nucleot[des along the inside of the L suggests that the presentation of these nucleotides by the tRNA structure to the synthetase is preserved from one tRNA sequence framework to another, The recent report of an unusual tercontact both ends of the L must rely on tiary iuteraction in E. coil tRNAcys that distinct domains for interaction. has a role in cysteine aminoacylation I° The formation of the L is facilitated challenged the view of the tRNA strucby base stacking and by tertiary hydro- ture as a passive scaffold for synthetase gen interactions between the conserved recognition. While all other cytoplasmic and semiconserved nucleotides. The tRNAs contain a purine at position 15 in acceptor stem stacks with the T¥C the D loop and a complementary pyrimstem to form one arm of the L, while the idine at position 48 in the variable loop, D stem and the anticodon stem stack to which establish a tertiary interaction form the other arm of the L. The con- known as the Levitt pair, E. coil tRNAcys served and semiconserved nucleotides has a G15 and a G48. Analyses with are concentrated in the D and the T~C chemical probes and structural modelloops, and contribute to the 'outside' ing show that G15 and G48 establish a corner of the L. The 'inside' of the L tertiary interaction through the exoconsists o[ the acceptor helix, the D cylic N2 as the hydrogen donor and the stem and the anticodon stem and loop, ring N3 as the acceptor. This tertiary and is the proposed primary site for interaction is different from the trasynthetase recognition 3. The co-crystal ditional tertiary interaction in a Levitt structure of Escherichia coli glutamine base pair, which would involve the N1 tRNA synthetase with tRNA~h', and of hydrogen as the donor and the 06 as yeast aspartyl tRNA synthetase with the acceptor. In E. coil tRNAcys, substitRNA.~p, provided evidence for tRNA- tution of G15:G48 to G15:C48 or synthetase interaction along the inside C15 : G48 virtually eliminates aminoacylof the L~ . Substitutions of specific ation, whereas creation of G15 • U48 or © 1993,ElsevierSciencePublishers. (UK) 0968--0004/93/$06.00
TIBS 1 8 - OCTOBER 1 9 9 3
U15 : G48 retains partial aminoacy[ation base pair of the acceptor stem to allow shortening the distance between the with cysteine. The dependence on the the ami[llo acid attachment site to turn two ends of the L. While the tRNA bendnature of the nucleotides that contrib- towards the inside of the L (Ref. 3). ing hypothesis remains to be tested by ute to the tertiary interaction contrasts Further, the anticodon loop of tRNAc'l" the crystal structure of tRNAcy', and the with that of the Levitt base pair. studies is altered so that the anticodon triplet co-crystal structure with cysteine tRNA of yeast tRNAPhe (which contains a is splayed out in order to specifically synthetase, it is to be noted that, in Levitt base pair) show that alterations contact the synthetase s. The cysteine the absence of this unique G15:G48 within the confines of base com- enzyme is related to the ,h]rosine and tertiary interaction, the cysteine enzyme plementarity do not ~ignific.~mtly affect ~uta~ine enzymes in that they all use fails to aminoacylate a variant of yeast the amino-terminal domain of the tRNAAsp that contains the anticodon aminoacylationu. The unusual GI5 : G48 tertiary interac- nuc]eotide-binding fold for amino acid and discriminator base of tRNAcy' (Y-M. tion is manifested by a structural con- altachment '~ and the carboxy-terminal Hou, unpublished). Clearly, the tertiary straint that projects the sugar- domain for anticodon recognition. The structure of tRNA plays a much more phosphate backbone of G15 away from compacl~ess of the cysteine enzyme active role in synthetase recognition the center of the L. This resutts in dis- arises from elimination of connective than was previously realized. Thus, ruption of the base-stacking interaction p o l ~ e p t i d e s in the amino-terminal between the anticodon and the amino that is normally observed between lee domain and a shortened carboxy-ter- acid attachment site that frame the purine rings of G15 and AI4 in the D minal domain ~. Assuming that the cys- inside of the L can lie structural signals loop. The GI5 of tRNAcy~ therefore pro- teine enzyme is a sphere, its diameter that control precision in the developtrudes into a position unique [Tom will be approximately 60 A or less ment of the genetic code. the position of the corresponding (P. Schimmel, pets. commun.). This nucleotide in all other tRNAs. Additinnal requires that either IRNATM bends in- Acknowledgements l thank P. Schimmel for reading the studies suggest that the defective wards, or that the enzyme is elongated G15 : C48 and C15 : G48 mutants shift the to span the distance from the anticodon manuscript and providing critical comments. This work was funded in part by sugar-phosphate backbone at position to the acceptor end. The protrusion of G15 in E. coil Grant GM47935-01 from the NIH and a 15 away from the wild-type position, whereas the G15 : U48 and UI5 : G48 tR.~g~-~ may induce tRNA to bend grant from Human Frontier Science mutants that retain partial aminoacy[- inwards. Because of the intimate link Program. ation align their nucleotide at position belween G15 and G48, and because of 15 with that of the wild type t°. These lee backbone cormection of G48 to the References I Rich, A. and Raj Bhandhary, U. L. (1976) Annu. studies illustrate the ability of E. coli cys- 3:end of the acceptor helix, the proRev. Biochern. 45, 805-860 teine tRNA synthetase to distinguish the trusion of GI5 is comparable to pulling 2 Musier-Forsyth,K. and Schimmel, P. (1993) the hinge of a right angle away from the details of tRNA structural variations. FASEB J. 7,282-289 Why does E. coil cysteine tRNA syn- center so as to bend the acceptor helix 3 Rich, A. and Schimmel, P. R. (19771 Nucleic Acids Res. 4, 1649-1664 thetase recruit a feature of the tRNA towards the anticodon helix, and thus tertiary structure as a means of tRNA discrimination? This enzyme differ~mioo arid atlarhmcnt entiates tRNAs based on f interactions with the anti0 0 codon triplet and the so0 called 'discriminator base' • Amino acid attachment that is adjacent to the tOO n loop T~PCstem 70 f amino acid attachment Accvl~or helix site r'. However, the cysteine enzyme is the smallH H 48k~-'~ Acceptor helix H T~C loop est monomeric synthetase in the synthetase family, being 461 residues long u. •@ oO : Z ~ ,.o ,I,I,I,I,,I, oo ~ aH Variahle h,,,p The smaller but dimeric • • H • 30H411 ty~'osine tRNA synthetase e-e ~ e---e Variable loop 0 @ (334 amino acids per _~O.-a4u* • • polypeptide chain) recog0 e nizes the anticodon and O ~ nticodontriplet the acceptor end of tRNATyr, but it most probAnlk'odontriplet ably achieves this by binding tRNATx' across the dimer interface ~4. The Figure 1 larger and monomeric The clo~erfe~ ,~1~.; a~ul ~ L-shaped (right) tRNA structures in the framework of E. colitRNACyhwhich glutamine tRNA synthetase has a~ a~c-..~g~, ~i~.----------------~/GCA. Each nucleotide is indicated by a circle, and the numbering is according to t h ~ a d ~ e ~ f ~ ~east tRNA~- (•ef. 16). Open circles represent the conserved and semiconserved (551 amino acids) contacts n~c[eGtJGes. ~ s.%=-g~.JG15 in the D loop and G48 in the variable loop establish an unusual tertiary both ends of tRNAc'l", but interact./,a~, i~ E_ oe~7t~'~.~~-~. with disruption of the last 363
"11BS 1 8 -
4 Rould, M, A., Perona, J. J., $611,D. and Steitz, T. A. (1989) Science 246, 1135-1142 5 Rould, M, A., Perona, J. J. and Steitz, T. A. (1991) Nature 352,213-218 6 Ruff, M. et al. (1991) Science 252, 1682-1689 7 .lahn, M., Rogers, M. J. and $611,D, (1991) Nature 352,258-260 8 POtz,J., Puglisi, J. D., Florentz, C. and Gieg6, R. (1991) Science 252, 1696-1699 9 P~tz, J., Puglisi, J. D., Florentz, C. and Gieg~.,R. (1993) EMBOJ. 12, 2944-2957 10 Hou, Y-M., Westhof, E. and Gieg6, R. (1993)
Proc. Nat/Acad. Sci. USA 90, 6776-6780 11 Sampson, J. R., DiRenzo,A, B., Behlen, L. S. and Uhlenbeck, O. C. (1990) Biochemistry 29, 2523-2532 12 Pallanck, L., Li, S. and Schulman, L. H. (1992) .I. Biol. Chem. 267, 7221-7223 13 Hou, Y-M., Shiba, K., Mottes, C. and Schimme5 P. (1991) Proc. Natl Acad. Sci. USA 88, 976-980 14 Carter, P., Bedouelle, H. and Winter, G. (1986) Proc. Nat/Acad. ScL USA 83, 1189-1192 15 Rossman, M. G., Liljas, A., Branden, C. I. and
OCTOBER 1993
Banaszak, L. (1976) in The Enzymes (Boyer, P. D., ed.), pp. 62-102, Academic Press 16 Sprinzl, M. et al. (1991) Nucleic Acids Res. 17, rl-r172
YA-MING HOU Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Bluemle Life Sciences Building, 233 South lOth Street, PA 19107, USA.
OBITUARY William Fields Harrington died on 31 October 1992, at the age of 72. At the time of his death he held the Henry Waiters Professorship of Biology at the Johns Hopkins University. This memoir was written on the occasion of a Memorial Symposium that was held at Johns Hopkins on 10-12 June 1993, at which his life and work were honored and celebrated. What follows is a brief tribute and remembrance by a few of his many friends and colleagues. Bill was born in Seattle on 25 September 1920, and attended public schools there. Following service in the Marine Corps at the end of World War II, he completed his undergraduate education at the University of California at Berkeley, He then entered graduate school at the same institution, where he carried out his studies for the PhD in the laboratory of Howard $chachman. His work there resulted in several outstanding papers that dealt primarily with the theory and practice of analytical ultracentrifugation and the structure and subunits of tobacco mosaic virus. His years at Berkeley initiated his lifelong passion for physical biochemistry and kindled his desire to apply this approach to the complicated problems of macromolecular structure, assembly and function. This conceptual progression culminated in the molecular studies of muscle structure and function that dominated much of his scientific life. After a year of postdoctoral work in the Department of Colloid Science at Cambridge University, England, Bill reached a major turning point when he took up a second postdoctoral fellowship at the Carlsberg Laboratory in Copenhagen, which, under Kai Linderstrom-Lang, had become the
364
\
\
most exciting place in the world in which to study protein physical chemistry. The laboratory was permeated by joyous scientific discussions, music and cigar smoke (Linderstrom-Lang was an accomplished violinist and inveterate cigar smoker), and almost all of the American visitors joined in by taking up both an instrument and an after-dinner cigar. Bill's instrument was the cello, and he enjoyed impromptu music sessions with his wife Inge, John and Charlotte Schellman, Chris and Flossie Anfinsen, and many others. The amateur status of the group is illustrated by the visit that Bill and Chris made to a music store to buy a Mozart quartet
containing no sharps or flats! Bill already possessed the qualities that made him such an outstanding colleague all his life: intelligence, humor, and an almost unique warmth and devotion to science. These characteristics, his background from the Berkeley years, and the fact that he soon discovered the analytical ultracentrifuge in the basement, instantly made Bill the local expert on macromolecular transport properties, and placed him at the center of many important scientific discussions and collaborations. In 1954 Bill left Copenhagen to take up his first academic position at Iowa State University. A year later he found that he could not resist the invitation of Chris Anfinsen to join his laboratory at the NIH, and it was here that he met Peter yon Hippel, Wayne Kielley and Elemer Mihalyi, among others, and began work on fibrous proteins, which started with collagen but soon moved on to myosin and ultimately to the study of full-blown muscle complexes. The NIH period saw the start of his use of proteolytic enzymes to probe the secondary structure of fibrous proteins, as well as his interest in helix-coil interconversion reactions in such systems. These approaches led him to study the formation and structure of the collagen triple helix, and ultimately to the concept of the hinge region within the myosin rod, which he suggested might undergo ATP-driven c~-helix ~- random coil transitions as part of the forcegenerating mechanism of muscle contraction. By 1960 many institutions had recognized Bill Harrington as one of the nation's foremost young physical biochemists. Mter considering many academic offers, Bill accepted a professor-
© 1993, Elsevier Science Publishers, (UK) 6968-0004/93/$06.00
of indefinitely extended polypeptide chains rather than discrete subunits linked partly by end-to-end interactions. In separate discussions of myosin and tropomyosin, no mention is made of any similarity to keratins. It would seem to be time for textbooks to present a unified discussion of the coiled coil as a distinct and widely occurring protein structure and to describe accurately the organization of proteins, including keratins, in which coiled coils occur.
References 1 Fuchs, E. et at. (1987) Curt. Top. Oev. Biol. 22, 5-34 2 Crick, F. C. (1953) Acta Cryst. 6, 689-697 3 Fraser,R. D. B. and MacRae,T. P. (1973) Conformation in Ifibrous Proteins, AcademicPress 4 Parry, D. A. D. (1990) in Cellular and Molecular Biology of Intermediate Filaments (Goldman,
The elucidation of the three-dimensional tRNA structure as an 'L' shape that folds from a cloverleaf secondary structure has an interesting outcome ~ (Fig. 1). The anticodon triplet and the amino acid attachment site are located at the opposite ends of the L, separated by about 75 A. In living cells, the connection that determines the relationship between amino acid attachment and the anticodon triplet is established by the 20 aminoacyl tRNA synthetases, one for each amino acid. This connection underlies the principles of the genetic code, where each of the 64 codon triplets that corresponds to a particular amino acid or stop instruction during protein synthesis is interpreted by tRNAs through nucleic acid base-pairing to the anticodon triplets. We have gained new insights into the question of how aminoacyl tRNA synthetases recognize tRNAs, in part because many synthetases have been found to specifically aminoacylate RNA oligonucleotides that derive from the acceptor stem sequences of the cognate tRNAs~. Because the acceptor stem is proximal to the amino acid attachment site, recognition by the synthetase can probably be accommodated by the same protein domain as interacts with the amino acid. However, a fraction of these synthetases also recognize the anticodon triplet for aminoacylation. The distal separation between the anticodon and the amino acid attachment site implies that the synthetases that
362
R. 0. and Steinert, P. M., eds), pp. 175-204, Plenum Press 5 Steinert, P. M. and Roop, D. R. (1988) Annu. Rev. Biochem. 57,593-625 6 Stewart, M. (1993) C~rr. Opin. Cell Biol, 5, 3-11 7 Cohen, C. and Parry, D. A. D. (1986) Trends Biochem. Sci. 11, 245-248 80'Shea, E. K., Klemm, J. D., Kim, P. S. and AIber, T. (1991) Science 254, 539-544 9 Conway, J. F. and Parry, D. A. D. (1991) Int. 1 Biol. Macremol, 13, 14-16 10 Lovejoy, 8. et al. (1993) Science 259, 1288-1293 11 Hatzfeld, M. and Weber, K. (1990) J. Ceil Biol. 110, 1199-1210 12 Steinert, P. M. (1990) J. Biol. Chem. 265, 8766-8774 13 Coulombe, P. A. and Fuchs, E. (1990) J. Cell Biol. 111, 153-169 14 Steinert, P. M. et al. (1991) Int. J. Biol. MacromoL 13, 130-139 15 Fraser, R. D. B., MacRae, T. P. and Parry, D. A. D. (1990)in Cellular and Molecular Biology of intermediate Filaments (Goldman, R. D. and Steinert, P. M., eds), pp, 205-231, Plenum Press
The tertiary structure of tRNA and the development of genetic code
16 Aebi, U., Fowler, W. E., Rew, P. and Sun, T. (1983) J. Ceil Biol. 97, 1131-1143 17 Engel, A., Eichner, E. and Aebi, U. (1985) J. Ultrastruct. Res. 90, 323-335 18 Steinert, P. M. (1991) J. Struct. Biol. 107, 157-174 19 Geisler, H., SchOnemann,J. and Weber, K. (1992) Eur. J. Biochem. 206, 841--852 20 Steven, A. C. (1990) in Cellular and Molecular Biology of Intermediate Filaments (Goldman, R. D. and Steinert, P. M., eds), pp. 233-263, Plenum Press 21 Lehninger, A. L., Nelson, D. L. and Cox, M. M. (1993) Principles of Biochemistry (2rid edn), Worth 22 Zubay, G. (1993) Biochemistry (3rd edn), Wm C. Brown 23 Matthews, C. K. and Van Holde, K. E, (1990) Biochemistry, Benjamin/Cummings
JEFFREY A. COHLBERG Department of Chemistry and Biochemistry, California State University, 1250 Bellflower Bird, Long Beach, CA 90840-0115, USA.
nucleotides that are in contact with the synthetase significantly decrease the catalytic efficiency of aminoacylation, and transfer of these nucleotides to a different tRNA sequence framework confers recognition of the latter by the synthetase 7-9, The ability to change tRNA identity by switching defined nucleot[des along the inside of the L suggests that the presentation of these nucleotides by the tRNA structure to the synthetase is preserved from one tRNA sequence framework to another, The recent report of an unusual tercontact both ends of the L must rely on tiary iuteraction in E. coil tRNAcys that distinct domains for interaction. has a role in cysteine aminoacylation I° The formation of the L is facilitated challenged the view of the tRNA strucby base stacking and by tertiary hydro- ture as a passive scaffold for synthetase gen interactions between the conserved recognition. While all other cytoplasmic and semiconserved nucleotides. The tRNAs contain a purine at position 15 in acceptor stem stacks with the T¥C the D loop and a complementary pyrimstem to form one arm of the L, while the idine at position 48 in the variable loop, D stem and the anticodon stem stack to which establish a tertiary interaction form the other arm of the L. The con- known as the Levitt pair, E. coil tRNAcys served and semiconserved nucleotides has a G15 and a G48. Analyses with are concentrated in the D and the T~C chemical probes and structural modelloops, and contribute to the 'outside' ing show that G15 and G48 establish a corner of the L. The 'inside' of the L tertiary interaction through the exoconsists o[ the acceptor helix, the D cylic N2 as the hydrogen donor and the stem and the anticodon stem and loop, ring N3 as the acceptor. This tertiary and is the proposed primary site for interaction is different from the trasynthetase recognition 3. The co-crystal ditional tertiary interaction in a Levitt structure of Escherichia coli glutamine base pair, which would involve the N1 tRNA synthetase with tRNA~h', and of hydrogen as the donor and the 06 as yeast aspartyl tRNA synthetase with the acceptor. In E. coil tRNAcys, substitRNA.~p, provided evidence for tRNA- tution of G15:G48 to G15:C48 or synthetase interaction along the inside C15 : G48 virtually eliminates aminoacylof the L~ . Substitutions of specific ation, whereas creation of G15 • U48 or © 1993,ElsevierSciencePublishers. (UK) 0968--0004/93/$06.00
TIBS 1 8 - OCTOBER 1 9 9 3
U15 : G48 retains partial aminoacy[ation base pair of the acceptor stem to allow shortening the distance between the with cysteine. The dependence on the the ami[llo acid attachment site to turn two ends of the L. While the tRNA bendnature of the nucleotides that contrib- towards the inside of the L (Ref. 3). ing hypothesis remains to be tested by ute to the tertiary interaction contrasts Further, the anticodon loop of tRNAc'l" the crystal structure of tRNAcy', and the with that of the Levitt base pair. studies is altered so that the anticodon triplet co-crystal structure with cysteine tRNA of yeast tRNAPhe (which contains a is splayed out in order to specifically synthetase, it is to be noted that, in Levitt base pair) show that alterations contact the synthetase s. The cysteine the absence of this unique G15:G48 within the confines of base com- enzyme is related to the ,h]rosine and tertiary interaction, the cysteine enzyme plementarity do not ~ignific.~mtly affect ~uta~ine enzymes in that they all use fails to aminoacylate a variant of yeast the amino-terminal domain of the tRNAAsp that contains the anticodon aminoacylationu. The unusual GI5 : G48 tertiary interac- nuc]eotide-binding fold for amino acid and discriminator base of tRNAcy' (Y-M. tion is manifested by a structural con- altachment '~ and the carboxy-terminal Hou, unpublished). Clearly, the tertiary straint that projects the sugar- domain for anticodon recognition. The structure of tRNA plays a much more phosphate backbone of G15 away from compacl~ess of the cysteine enzyme active role in synthetase recognition the center of the L. This resutts in dis- arises from elimination of connective than was previously realized. Thus, ruption of the base-stacking interaction p o l ~ e p t i d e s in the amino-terminal between the anticodon and the amino that is normally observed between lee domain and a shortened carboxy-ter- acid attachment site that frame the purine rings of G15 and AI4 in the D minal domain ~. Assuming that the cys- inside of the L can lie structural signals loop. The GI5 of tRNAcy~ therefore pro- teine enzyme is a sphere, its diameter that control precision in the developtrudes into a position unique [Tom will be approximately 60 A or less ment of the genetic code. the position of the corresponding (P. Schimmel, pets. commun.). This nucleotide in all other tRNAs. Additinnal requires that either IRNATM bends in- Acknowledgements l thank P. Schimmel for reading the studies suggest that the defective wards, or that the enzyme is elongated G15 : C48 and C15 : G48 mutants shift the to span the distance from the anticodon manuscript and providing critical comments. This work was funded in part by sugar-phosphate backbone at position to the acceptor end. The protrusion of G15 in E. coil Grant GM47935-01 from the NIH and a 15 away from the wild-type position, whereas the G15 : U48 and UI5 : G48 tR.~g~-~ may induce tRNA to bend grant from Human Frontier Science mutants that retain partial aminoacy[- inwards. Because of the intimate link Program. ation align their nucleotide at position belween G15 and G48, and because of 15 with that of the wild type t°. These lee backbone cormection of G48 to the References I Rich, A. and Raj Bhandhary, U. L. (1976) Annu. studies illustrate the ability of E. coli cys- 3:end of the acceptor helix, the proRev. Biochern. 45, 805-860 teine tRNA synthetase to distinguish the trusion of GI5 is comparable to pulling 2 Musier-Forsyth,K. and Schimmel, P. (1993) the hinge of a right angle away from the details of tRNA structural variations. FASEB J. 7,282-289 Why does E. coil cysteine tRNA syn- center so as to bend the acceptor helix 3 Rich, A. and Schimmel, P. R. (19771 Nucleic Acids Res. 4, 1649-1664 thetase recruit a feature of the tRNA towards the anticodon helix, and thus tertiary structure as a means of tRNA discrimination? This enzyme differ~mioo arid atlarhmcnt entiates tRNAs based on f interactions with the anti0 0 codon triplet and the so0 called 'discriminator base' • Amino acid attachment that is adjacent to the tOO n loop T~PCstem 70 f amino acid attachment Accvl~or helix site r'. However, the cysteine enzyme is the smallH H 48k~-'~ Acceptor helix H T~C loop est monomeric synthetase in the synthetase family, being 461 residues long u. •@ oO : Z ~ ,.o ,I,I,I,I,,I, oo ~ aH Variahle h,,,p The smaller but dimeric • • H • 30H411 ty~'osine tRNA synthetase e-e ~ e---e Variable loop 0 @ (334 amino acids per _~O.-a4u* • • polypeptide chain) recog0 e nizes the anticodon and O ~ nticodontriplet the acceptor end of tRNATyr, but it most probAnlk'odontriplet ably achieves this by binding tRNATx' across the dimer interface ~4. The Figure 1 larger and monomeric The clo~erfe~ ,~1~.; a~ul ~ L-shaped (right) tRNA structures in the framework of E. colitRNACyhwhich glutamine tRNA synthetase has a~ a~c-..~g~, ~i~.----------------~/GCA. Each nucleotide is indicated by a circle, and the numbering is according to t h ~ a d ~ e ~ f ~ ~east tRNA~- (•ef. 16). Open circles represent the conserved and semiconserved (551 amino acids) contacts n~c[eGtJGes. ~ s.%=-g~.JG15 in the D loop and G48 in the variable loop establish an unusual tertiary both ends of tRNAc'l", but interact./,a~, i~ E_ oe~7t~'~.~~-~. with disruption of the last 363
"11BS 1 8 -
4 Rould, M, A., Perona, J. J., $611,D. and Steitz, T. A. (1989) Science 246, 1135-1142 5 Rould, M, A., Perona, J. J. and Steitz, T. A. (1991) Nature 352,213-218 6 Ruff, M. et al. (1991) Science 252, 1682-1689 7 .lahn, M., Rogers, M. J. and $611,D, (1991) Nature 352,258-260 8 POtz,J., Puglisi, J. D., Florentz, C. and Gieg6, R. (1991) Science 252, 1696-1699 9 P~tz, J., Puglisi, J. D., Florentz, C. and Gieg~.,R. (1993) EMBOJ. 12, 2944-2957 10 Hou, Y-M., Westhof, E. and Gieg6, R. (1993)
Proc. Nat/Acad. Sci. USA 90, 6776-6780 11 Sampson, J. R., DiRenzo,A, B., Behlen, L. S. and Uhlenbeck, O. C. (1990) Biochemistry 29, 2523-2532 12 Pallanck, L., Li, S. and Schulman, L. H. (1992) .I. Biol. Chem. 267, 7221-7223 13 Hou, Y-M., Shiba, K., Mottes, C. and Schimme5 P. (1991) Proc. Natl Acad. Sci. USA 88, 976-980 14 Carter, P., Bedouelle, H. and Winter, G. (1986) Proc. Nat/Acad. ScL USA 83, 1189-1192 15 Rossman, M. G., Liljas, A., Branden, C. I. and
OCTOBER 1993
Banaszak, L. (1976) in The Enzymes (Boyer, P. D., ed.), pp. 62-102, Academic Press 16 Sprinzl, M. et al. (1991) Nucleic Acids Res. 17, rl-r172
YA-MING HOU Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Bluemle Life Sciences Building, 233 South lOth Street, PA 19107, USA.
OBITUARY William Fields Harrington died on 31 October 1992, at the age of 72. At the time of his death he held the Henry Waiters Professorship of Biology at the Johns Hopkins University. This memoir was written on the occasion of a Memorial Symposium that was held at Johns Hopkins on 10-12 June 1993, at which his life and work were honored and celebrated. What follows is a brief tribute and remembrance by a few of his many friends and colleagues. Bill was born in Seattle on 25 September 1920, and attended public schools there. Following service in the Marine Corps at the end of World War II, he completed his undergraduate education at the University of California at Berkeley, He then entered graduate school at the same institution, where he carried out his studies for the PhD in the laboratory of Howard $chachman. His work there resulted in several outstanding papers that dealt primarily with the theory and practice of analytical ultracentrifugation and the structure and subunits of tobacco mosaic virus. His years at Berkeley initiated his lifelong passion for physical biochemistry and kindled his desire to apply this approach to the complicated problems of macromolecular structure, assembly and function. This conceptual progression culminated in the molecular studies of muscle structure and function that dominated much of his scientific life. After a year of postdoctoral work in the Department of Colloid Science at Cambridge University, England, Bill reached a major turning point when he took up a second postdoctoral fellowship at the Carlsberg Laboratory in Copenhagen, which, under Kai Linderstrom-Lang, had become the
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most exciting place in the world in which to study protein physical chemistry. The laboratory was permeated by joyous scientific discussions, music and cigar smoke (Linderstrom-Lang was an accomplished violinist and inveterate cigar smoker), and almost all of the American visitors joined in by taking up both an instrument and an after-dinner cigar. Bill's instrument was the cello, and he enjoyed impromptu music sessions with his wife Inge, John and Charlotte Schellman, Chris and Flossie Anfinsen, and many others. The amateur status of the group is illustrated by the visit that Bill and Chris made to a music store to buy a Mozart quartet
containing no sharps or flats! Bill already possessed the qualities that made him such an outstanding colleague all his life: intelligence, humor, and an almost unique warmth and devotion to science. These characteristics, his background from the Berkeley years, and the fact that he soon discovered the analytical ultracentrifuge in the basement, instantly made Bill the local expert on macromolecular transport properties, and placed him at the center of many important scientific discussions and collaborations. In 1954 Bill left Copenhagen to take up his first academic position at Iowa State University. A year later he found that he could not resist the invitation of Chris Anfinsen to join his laboratory at the NIH, and it was here that he met Peter yon Hippel, Wayne Kielley and Elemer Mihalyi, among others, and began work on fibrous proteins, which started with collagen but soon moved on to myosin and ultimately to the study of full-blown muscle complexes. The NIH period saw the start of his use of proteolytic enzymes to probe the secondary structure of fibrous proteins, as well as his interest in helix-coil interconversion reactions in such systems. These approaches led him to study the formation and structure of the collagen triple helix, and ultimately to the concept of the hinge region within the myosin rod, which he suggested might undergo ATP-driven c~-helix ~- random coil transitions as part of the forcegenerating mechanism of muscle contraction. By 1960 many institutions had recognized Bill Harrington as one of the nation's foremost young physical biochemists. Mter considering many academic offers, Bill accepted a professor-
© 1993, Elsevier Science Publishers, (UK) 6968-0004/93/$06.00