CF Francklyn - Academia.edu (original) (raw)

Papers by CF Francklyn

Biochemistry, 2008

DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critic... more DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critical roles in the transformation of genetic information from DNA to RNA to protein. DNA polymerases carry out replication and collaborate in the repair of the genome, while ARSs provide aminoacylated tRNA precursors for protein synthesis. Enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high levels of discrimination with the assistance of proofreading mechanisms. Here, the fidelity preservation mechanisms in these two important systems are reviewed, and similar features highlighted. Among the noteworthy features common to both DNA polymerases and ARSs are the use of multidomain architectures that segregate synthetic and proofreading functions into discrete domains; the use of induced fit to enhance binding selectivity; the imposition of fidelity at the level of chemistry; and the use of post-chemistry error correction mechanisms to hydrolyze incorrect products in a discrete editing domain. These latter mechanisms further share the common property that error correction involves the translocation of mis-incorporated products from the synthetic to the editing site, and that the accuracy of the process may be influenced by the rates of translocation in either direction. Fidelity control in both families can thus be said to rely on multiple elementary steps, each with its contribution to overall fidelity. The summed contribution of these “kinetic checkpoints” provides the high observed overall accuracy of DNA replication and aminoacylation.

Journal of Biological Chemistry, 2009

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein syn... more Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5-O-[N-(L-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.

We have used a secondary site suppression approach to investigate the basis of tRNA selection by ... more We have used a secondary site suppression approach to investigate the basis of tRNA selection by the E. coli histidyl-tRNA synthetase. This enzyme recognizes a unique G-1:C73 base pair located in the acceptor stems of prokaryotic histidyl-tRNAs. A genetic selection system was constructed in which growth on glycerol was dependent on histidine specific amber suppression of a triose phosphate isomerase (tpi) gene containing an amber codon at His 95. Three independent revertants linked to hisS were isolated and sequenced, and the resulting mutant proteins were characterized biochemically. These studies are interpreted in light of the x-ray structure of the E. coli HisRS adenylate complex, and show that the C-terminal domain and its interactions with the catalytic domain play a biologically significant role in tRNA selection.

Nucleic acids symposium series, 1995

Cancer Research, 2013

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In mammalian systems,... more Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In mammalian systems, several aminoacyl-tRNA synthetases are known to have distinct non-canonical functionality. For the first time, we demonstrate that human threonyl-tRNA synthetase (TARS) induces angiogenesis through endothelial cell signaling, and this induction can be blocked by the borrelidin derivative compound BC194. Upon stimulation with vascular endothelial growth factor (VEGF) or tumor necrosis factor-alpha (TNF-α), human endothelial cells secrete TARS into the extracellular space. Extracellular TARS induces and BC194 inhibits angiogenesis in both in vitro tube formation assays and in vivo chick chorioallantoic membrane assays. Furthermore, the anti-angiogenic effects of BC194 are specific to its inhibition of TARS angiogenic activity rather than an indirect stress response. Additional evidence supports a stimulatory role for TARS in endothelial cell migration as a mechanism for its angiogenic activity. Taken together, these findings reveal a previously undefined function for TARS as an angiogenesis inducer stimulated by hypoxic and inflammatory signals. Detecting or intervening in the non-canonical extracellular TARS signaling pathway may thus provide a novel means of diagnosing or treating angiogenesis and inflammatory responses involved in cancer. Citation Format: Tamara Williams, Adam Mirando, Christopher Francklyn, Karen M. Lounsbury. A novel function for threonyl-tRNA synthetase as a stimulator of angiogenesis and endothelial cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2013-3897

Trends in Biochemical Sciences, 1989

Proceedings of the National Academy of Sciences, 2003

Proceedings of the National Academy of Sciences, 1990

The major determinant for the identity of alanine tRNAs is a single base pair in the acceptor hel... more The major determinant for the identity of alanine tRNAs is a single base pair in the acceptor helix that is proximal to the site of amino acid attachment. A 7-base-pair microhelix that recreates the acceptor helix can be charged with alanine. No other examples of charging of small helices with specific amino acids have been reported, to our knowledge. We show here that a 13-base-pair and an 8-base-pair hairpin helix that reconstruct a domain and subdomain, respectively, of histidine tRNAs can be charged with histidine. We also show that transplantation of a base pair that is unique to histidine tRNAs is sufficient to consider histidine acceptance on a domain and subdomain of alanine tRNA. Both alanine and histidine aminoacyl-tRNA synthetases retain specficity for their cognate synthetic substrates. Alanine-and histidine-specific microhelices may resemble a system that arose early in the evolution of charging and coding.

Proceedings of the National Academy of Sciences, 1999

In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases... more In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways. Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase (HisG). Prediction of the function of HisZ in Lactococcus lactis was assisted by comparative genomics, a technique that revealed a link between the presence or the absence of HisZ and a systematic variation in the length of the HisG polypeptide. HisZ is required for histidine prototrophy, and three other lines of evidence support the direct involvement of HisZ in the transferase function. (i) Genetic experiments demonstrate that complementation of an in-frame deletion of HisG from Escherichia coli (which does not possess HisZ) requires both HisG and HisZ from L. lactis. (ii) Coelution of HisG and HisZ during affinity chromatography provides evidence of direct physical interaction. (iii) Both HisG and HisZ are required for catalysis of the ATP phosphoribosyltransferase reaction. This observation of a common protein domain linking amino acid biosynthesis and protein synthesis implies an early connection between the biosynthesis of amino acids and proteins. MATERIALS AND METHODS Materials. Escherichia coli 16S͞23S RNA is from Boehringer Mannheim. The hairpin ribozyme RNA was a gift from J. Burke and J. Heckman (University of Vermont, Burlington). HT-Tuffryn (0.45-m porosity) membranes were obtained The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Nucleic Acids Research, 1999

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfam... more The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the λ repressor operator O R 1 and two model sequence targets containing imperfect versions of the 5′-TAAT-3′ consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.

Journal of Molecular Biology, 1998

The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation... more The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation mechanism in which the level of histidyl-tRNA serves as a key sensor of the intracellular histidine pool. Among the early observations that led to the formation of this model for Salmonella typhimurium were the identi®cation of mutants in the gene (hisS) encoding histidyl-tRNA synthetase. We report here the detailed biochemical characterization of ®ve of these S. typhimurium bradytrophic mutants isolated by selection for resistance to histidine analogs, including identi®cation of the deduced amino acid substitutions and determination of the resulting effects on the kinetics of adenylation and aminoacylation. Using the crystal structure of the closely related Escherichia coli histidyl-tRNA synthetase (HisRS) as a guide, two mutants were mapped to a highly conserved proline residue in motif 2 (P117S, P117Q), and were correlated with a ®vefold decrease in the k cat for the pyrophosphate exchange reaction, as well as a tenfold increase in the K m for tRNA in the aminoacylation reaction. Another mutant substitution (A302T) mapped to a residue adjacent to the histidine binding pocket, leading to a tenfold increase in K m for histidine in the pyrophosphate exchange reaction. The remaining two mutants (S167F, N254T) substitute residues in or directly adjacent to the hinge region, which joins the insertion domain between motif 2 and motif 3 to the catalytic core, and cause the K m for tRNA to increase four-to tenfold. The kinetic analysis of these mutants establishes a direct link between critical interactions within the active site of HisRS and regulation of histidine biosynthesis, and provides further evidence for the importance of local conformational changes during the catalytic cycle.

Journal of Molecular Evolution, 2000

In Lactococcus lactis there is a protein, HisZ, in the histidine-biosynthetic operon that exhibit... more In Lactococcus lactis there is a protein, HisZ, in the histidine-biosynthetic operon that exhibits significant sequence identity with histidyl-tRNA synthetase (HisRS) but does not aminoacylate tRNA. HisRS homologs that, like HisZ, cannot aminoacylate tRNA are represented in a highly divergent set of bacteria (including an aquificale, cyanobacteria, firmicutes, and proteobacteria), yet are missing from other bacteria, including mycrobacteria and certain proteobacteria. Phylogenetic analysis of the HisRS and HisRS-like family suggests that the HisZ proteins form a monophyletic group that attaches outside the predominant bacterial HisRS clade. These observations are consistent with a model in which the absences of HisZ from bacteria are due to its loss during evolution. It has recently been shown that HisZ from L. lactis binds to the ATP-PRPP transferase (HisG) and that both HisZ and HisG are required for catalyzing the first reaction in histidine biosynthesis. Phylogenetic analysis of HisG sequences shows conclusively that proteobacterial HisG and histidinol dehydrogenase (HisD) sequences are paraphyletic and that the partition of the Proteobacteria associated with the presence/absence of HisZ corresponds to that based on HisG and HisD paraphyly. Our results suggest that horizontal gene transfer played an important role in the evolution of the regulation of histidine biosynthesis.

Journal of Biological Chemistry, 2011

Four minimal (119-145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA s... more Four minimal (119-145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine activation as fusion proteins and after TEV cleavage, using the 32 PP i exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (ϳ0.1-0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered K m values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ϳ10 9 times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate amino acids. These fragments thus likely represent Urzymes (Ur ‫؍‬ primitive, original, earliest ؉ enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.

Journal of Biological Chemistry, 2013

Background: RNA World scenarios require high initial fidelity, greatly slowing lift-off. Results:... more Background: RNA World scenarios require high initial fidelity, greatly slowing lift-off. Results: Class I TrpRS and Class II HisRS Urzymes (120-130 residues) both acylate tRNAs ϳ10 6 times faster than the uncatalyzed peptide synthesis rate. Conclusion: Urzymes appear highly evolved, implying that they had even simpler ancestors. Significance: High Urzyme catalytic proficiencies imply that translation began in a Peptide⅐RNA World. We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120-130-residue Urzymes (Ur ‫؍‬ primitive plus enzyme) derived from Class I and Class II aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated amino acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both amino acid activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ϳ60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme⅐tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide⅐RNA development of rapid coding systems matching amino acids with anticodon trinucleotides.

… , M., Francklyn, C., and Cusack, S., Eds.) pp, 2005

... Edited by: Michael Ibba, Christopher Francklyn and Stephen Cusack. ... the 21stamino acid s... more ... Edited by: Michael Ibba, Christopher Francklyn and Stephen Cusack. ... the 21stamino acid since its specific incorporation into proteins isDNA-encoded and takes place cotranslationally at theribosome.1,2 Selenocysteine insertion, therefore, can be taken asa paradigmatic case ...

Journal of molecular biology, 1994

... 5. Leucyl-tRNA Synthetases Tommie L. Lincecum, Jr. and Susan A. Martinis. 6. Methionyl-tRNA S... more ... 5. Leucyl-tRNA Synthetases Tommie L. Lincecum, Jr. and Susan A. Martinis. 6. Methionyl-tRNA Synthetases Sylvain Blanquet, Thibaut Crépin, Yves Mechulam and Emmanuelle Schmitt. 7. Valyl-tRNA Synthetases Osamu Nureki and Shigeyuki Yokoyama. ...

Nucleic acids research, 1990

The preparation of fully protected dllsopropylamino/3-cyanoethyl ribonucleoside phosphoramidites ... more The preparation of fully protected dllsopropylamino/3-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity > 99.95% Is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-/Bu ...