Simon H. Chang

The corrected nucleotide sequence of yeast leucine transfer ribonucleic acid

Abstract The nucleotide sequence of “Renaturable” leucine transfer RNA from Bakers yeast has been re-investigated. The results showed that (i) this tRNA has a sequence of DCD at positions 19–21, (ii) it has an anticodon m 5 CAA and (iii) it has a pseudouridine at position 40.

The Crystal Structures of Eukaryotic Phosphofructokinases from Baker's Yeast and Rabbit Skeletal Muscle.

Phosphofructokinase 1 (PFK) is a multisubunit allosteric enzyme that catalyzes the principal regulatory step in glycolysis-the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by ATP. The activity of eukaryotic PFK is modulated by a number of effectors in response to the cells needs for energy and building blocks for biosynthesis. The crystal structures of eukaryotic PFKs-from Saccharomyces cerevisiae and rabbit skeletal muscle-demonstrate how successive gene duplications and fusion are reflected in the protein structure and how they allowed the evolution of new functionalities. The basic framework inherited from prokaryotes is conserved, and additional levels of structural and functional complexity have evolved around it. Analysis of protein-ligand complexes has shown how PFK is activated by fructose 2,6-bisphosphate (a powerful PFK effector found only in eukaryotes) and reveals a novel nucleotide binding site. Crystallographic results have been used as the basis for structure-based effector design.

Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp‐39 is replaced with asparagine, has a 60‐fold increase in affinity for NADP+ and a 1.5‐fold increase in k cat compared to wild‐type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263–267] and proposed that this part of ADH is close to the 2′‐phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala‐46 with an argine residue, an A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three‐dimensional structure of the NAD+‐binding region of ADH. The A46R mutant has a 2.5‐fold lower K m(app)NADP+ and a 3‐fold higher k cat with NADP+ compared to wild‐type ADH; binding of NAD+ to the mutant was unchanged and k cat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of k cat/K m of NAD+ to NADP+ is 85, over ten‐fold lower than that for wild‐type ADH. Our model of the 3D structure of the NAD+‐binding region of ADH shows that Ala‐46 is over 10 Å from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp‐39 and the 2′‐phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30‐fold lower K m(app)NADP+ and 8‐fold higher k cat with NADP+ as a cofactor compared to wild‐type ADH; binding of NAD+ to this double mutant was lowered by 5‐fold and k cat was increased by 1.5‐fold. As a result, k cat/K m for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; k cat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.

Nucleotide sequence of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene

The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced. The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis. The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes. A significant degree of homology exists when the deduced amino acid sequence of B. stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E. coli PFK-1. The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster.

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural analysis of nonradioactive rna by postlabeling: The primary structure of baker's yeast tRNACUALeu

Summary The sequence of tRNA CUA Leu from bakers yeast was determined by application of novel postlabeling methods. Oligonucleotides in complete and partial RNase digests (A, T 1 , and U 2 ) were resolved by chromatography on polyethyleneimine-cellulose thin layers. Nucleotide sequences were elucidated by NaIO 4 /phosphatase or snake venom phosphodiesterase/phosphatase digestion and tritium postlabeling. A ribose-methylated constituent (Gm) was identified by 5′-terminal [ 32 P]-labeling. tRNA CUA Leu is the first biological macromolecule to be sequenced according to a postlabeling scheme.

A fluorescent nucleoside from glutamic acid tRNA of Escherichia coli K 12

Abstract Glutamic acid tRNA from E. coli K 12 contains three minor nucleosides in the anticodon loop, namely, 2-methyladenosine, 5-methylaminomethyl-2-thioridine and a modified pyrimidine nucleoside, which is highly fluorescent. This tRNA has a chain length of 76 nucleotides and does not contain 7-methylguanosine or dihydrouridine as do the other nine sequenced E. coli tRNAs of chain length 76–77 nucleotides.

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom.

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.

Nucleotide sequence of "renaturable" leucine transfer ribonucleic acid.

“Renaturable” leucine transfer ribonucleic acid from baker’s yeast was found by Lindahl et al. [l] to be able to exist in a relatively stable denatured configuration which has only low amino acid acceptor activity. Incubation at 60’ for 5 min, followed by slow cooling in the presence of magnesium ion, was found to disrupt the incorrect cornformation and allow renaturation to -the native, biologically active structure. Although the physiological significance of the reversible denaturation process, if any, is not known, it is of interest to try to determine if there is a unique feature of the primary or secondary structure that would account for the stability of the second, inactive conformation. The “renaturable leucine tRNA (tRNAy)** was purified in our laboratory by sucessive chromatography on benzoylated DEAE cellulose and Sephadex G-100 columns [2]. We determined the nucleotide sequence of this tRNA [3] by Holley’s method [4]. In this paper, we would like to report our results and compare them with those recently reported by Kowalski et al. [S] . Purified tRNALeU was digested with pancreatic ribonuclease [2] .“The identity of the products from this digestion are shown in table 1. Sequence analyses

High-level expression of Bacillus stearothermophilus 6-phosphofructo-l-kinase in Escherichia coli

The 6-phosphofructo-1-kinase (PFK) gene from Bacillus stearothermophilus has been expressed at high levels in Escherichia coli. This expression has been demonstrated by complementation studies, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and PFK assays of cell extracts. A level of B. stearothermophilus PFK expression corresponding to 20% of the total extracted protein was calculated from densitometric scans of an SDS-polyacrylamide gel. The high level of recombinant gene expression will enable this laboratory to determine structure-function relationships in B. stearothermophilus PFK by the method of oligodeoxynucleotide-directed mutagenesis.