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L H Schulman

Publications and source records attributed to L H Schulman.

At least 19 recordsLinked to original sources
PubMed1994

Analysis of acceptor stem base pairing on tRNA(Trp) aminoacylation and function in vivo.

The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNA(Trp) was examined by complementation of an E. coli strain containing a temperature-sensitive tRNA(Trp) gene (trpTts) and by monitoring aminoacylation levels in vivo. All derivatives of tRNA(Trp) containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 degrees C). However, three acceptor stem derivatives (tRNA(Trp)/C1.G72, tRNA(Trp)/C2.G71, and tRNA(Trp)/A3.U70) required overexpression for growth at 42 degrees C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRNAs appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNA(UCATrp) derivatives. These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA(Trp). Substitution of the C1.A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNA(CCAfMet)/G73) with A1.U72, the base pair found in tRNA(Trp), or G1.C72 resulted in the conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo. However, changes to U1.A72 or C1.G72 in tRNA(CCAfMet)/G73 resulted in misaminoacylation and/or defects in translation. Our data indicate that the A1.U72 base pair is a context-dependent, negative identity element of tRNA(Trp).

Anticodon↗
PubMed1993

The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase.

We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1993

Two separate peptides in Escherichia coli methionyl-tRNA synthetase form the anticodon binding site for methionine tRNA.

The amino acid residues Asn391, Arg395, and Trp461 in methionyl-tRNA synthetase (MetRS) of Escherichia coli are involved in the anticodon-dependent recognition of its cognate tRNAs [Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225; Ghosh, G., Kim, H.Y., Demaret, J. P., Brunie, S., & Schulman, L.H. (1991) Biochemistry 30, 11767-11774]. While tryptophan at position 461 was shown to bind directly to the wobble base at position 34 in the tRNA(Met) anticodon, the role of residues 391-395 was not thoroughly explored. To gain further insight into the role of the 391-395 residues and nearby residues, appropriate mutations were analyzed for aminoacylation activity, as well as tRNA binding. Mutations of the phylogenetically conserved asparagine at position 391 increased the Km for aminoacylation of tRNA(Met) 18-40-fold. Further analysis using fluorescence titration indicated that the mutation affected initial complex formation, since the Kd for tRNA(Met) binding had increased at least 15-fold over wild type. Kinetic analysis of mutationally altered derivatives of MetRS with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interaction between the amino acid residue at position 391 and the U36 of the anticodon of tRNA(Met). In addition to position 391, position 387 was also found to affect tRNA(Met) binding and aminoacylation, indicating a possible significant role in interaction of the enzyme with the anticodon of tRNA(Met). These results indicate that the peptide segment containing residues 391-395 is involved in the direct recognition of the 3' end of the anticodon.

Acylation↗
PubMed1993

The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA(Asn).

A gel shift assay that distinguishes the aminoacylated form from the deacylated form of tRNAs was used to study the requirements for aminoacylation of Escherichia coli tRNA(Asn) in vivo. tRNA(Asn) derivatives containing single base changes in their anticodons or discriminator bases were constructed, and the extent of in vivo aminoacylation was determined directly. Substitution of U35 with C35 or U36 with C36 abolished aminoacylation of the tRNA. Substitution of G34 with C34 converted tRNA(Asn) into a lysine acceptor. Thus, each of the anticodon nucleotides are important for aminoacylation of tRNA(Asn). Substitution of discriminator base G73 with A73 affected the extent of aminoacylation in vivo indicating that the discriminator base also contributes to aminoacylation of tRNA(Asn).

Acylation↗
PubMed1992

The anticodon and discriminator base are major determinants of cysteine tRNA identity in vivo.

Mutants of the Escherichia coli initiator tRNA (tRNA(fMet)) have been used to examine the role of the anticodon and discriminator base in in vivo aminoacylation of tRNAs by cysteinyl-tRNA synthetase. Substitution of the methionine anticodon CAU with the cysteine anticodon GCA was found to allow initiation of protein synthesis by the mutant tRNA from a complementary initiation codon in a reporter protein. Sequencing of the protein revealed that cysteine comprised about half of the amino acid at the N terminus. An additional mutation, converting the discriminator base of tRNA(GCAfMet) from A73 to the base present in tRNA(Cys) (U73), resulted in a 6-fold increase in the amount of protein produced and insertion of greater than or equal to 90% cysteine in response to the complementary initiation codon. Substitution of C73 or G73 at the discriminator position led to insertion of little or no cysteine, indicating the importance of U73 for recognition of the tRNA by cysteinyl-tRNA synthetase. Single base changes in the anticodon of tRNA(GCAfMet) containing U73 from GCA to UCA, GUA, GCC, and GCG (changes underlined) eliminated or dramatically reduced cysteine insertion by the mutant initiator tRNA indicating that all three cysteine anticodon bases are essential for specific aminoacylation of the tRNA with cysteine in vivo.

Acylation↗
PubMed1992

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1991

Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225]. In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme. Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity. Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity. Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in kcat/Km for aminoacylation of tRNAfMet, with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate. In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km by 25,000-fold. The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E. coli having an altered chromosomal MetRS gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1991

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.

Adenosine Triphosphate↗
PubMed1991

Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo.

An assay based on the initiation of protein synthesis in Escherichia coli has been used to explore the role of the anticodon in tRNA identity in vivo. Mutations were introduced into the initiator tRNA to change the wild-type anticodon from CAU (methionine) to GAU (isoleucine), GAC (valine), and GAA (phenylalanine), where each derivative differs from the preceding by a single base change in the anticodon (underlined). These changes were sufficient to cause the mutant tRNAs to be aminoacylated by the corresponding aminoacyl-tRNA synthetases based on the amino acid inserted into protein from complementary initiation codons. Construction of additional single base anticodon variants (GUU, GGU, GCC, GUC, GCA, and UAA) showed that all three anticodon bases specify isoleucine and phenylalanine identity and that both the middle and the third anticodon bases are important for valine identity in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Initiation of in vivo protein synthesis with non-methionine amino acids.

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.

Amino Acid Sequence↗
PubMed1990

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1990

An anticodon change switches the identity of E. coli tRNA(mMet) from methionine to threonine.

Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases. In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E. coli threonyl-tRNA synthetase, derivatives of the E. coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity. Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount. These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.

Anticodon↗
PubMed1989

The anticodon contains a major element of the identity of arginine transfer RNAs.

The contribution of the anticodon to the discrimination between cognate and noncognate tRNAs by Escherichia coli Arg-tRNA synthetase has been investigated by in vitro synthesis and aminoacylation of elongator methionine tRNA (tRNA(mMet) mutants. Substitution of the Arg anticodon CCG for the Met anticodon CAU leads to a dramatic increase in Arg acceptance by tRNA(mMet). A nucleotide (A20) previously identified by others in the dihydrouridine loop of tRNA(Arg)s makes a smaller contribution to the conversion of tRNA(mMet) identity from Met to Arg. The combined anticodon and dihydrouridine loop mutations yield a tRNA(mMet) derivative that is aminoacylated with near-normal kinetics by the Arg-tRNA synthetase.

Anticodon↗
PubMed1988

Anticodon switching changes the identity of methionine and valine transfer RNAs.

The anticodon has previously been shown to play a role in recognition of certain transfer RNAs by aminoacyl-tRNA synthetases; however, the extent to which this sequence dictates tRNA identity is generally unknown. To investigate the contribution of the anticodon to the identity of Escherichia coli methionine and valine tRNAs, in vitro transcripts of these tRNAs were prepared that contained normal and interchanged anticodon sequences. Transcripts containing wild-type tRNA sequences were excellent substrates for their respective cognate aminoacyl-tRNA synthetases and were effectively discriminated against by a variety of noncognate enzymes. The mutant tRNAs produced by switching the anticodon sequences lost their original tRNA identity and assumed an identity corresponding to the acquired anticodon sequence. These results indicate that the anticodon contains sufficient information to distinguish methionine and valine tRNAs with high fidelity.

Anticodon↗
PubMed1987

Peptides at the tRNA binding site of the crystallizable monomeric form of E. coli methionyl-tRNA synthetase.

A protein affinity labeling derivative of E. coli tRNA(fMet) carrying lysine-reactive cross-linking groups has been covalently coupled to monomeric trypsin-modified E. coli methionyl-tRNA synthetase. The cross-linked tRNA-synthetase complex has been isolated by gel filtration, digested with trypsin, and the tRNA-bound peptides separated from the bulk of the free tryptic peptides by anion exchange chromatography. The bound peptides were released from the tRNA by cleavage of the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding three major peptides. These peptides were found to cochromatograph with three peptides of known sequence previously cross-linked to native methionyl-tRNA synthetase through lysine residues 402, 439 and 465. These results show that identical lysine residues are in close proximity to tRNA(fMet) bound to native dimeric methionyl-tRNA synthetase and to the crystallizable monomeric form of the enzyme, and indicate that cross-linking to the dimeric protein occurs on the occupied subunit of the 1:1 tRNA-synthetase complex.

Amino Acyl-tRNA Synthetases↗