Steven T. Gregory
- PhD SUNY Buffalo, 1992
- BA SUNY Buffalo, 1985
- CMB311 Introductory Biochemistry
- CMB311H Honors Introductory Biochemistry
- Mechanisms of antibiotic resistance
Protein synthesis by the ribosome is a structurally dynamic process consisting of a sequence of kinetically and structurally distinguishable steps. Many antibiotics act by perturbing the kinetics or the fidelity of these steps, in some instances by capturing the ribosome in non-productive conformations. Mutational alterations in RNA or protein components of the ribosome can confer antibiotic resistance, presumably by either distorting the conformation of the drug binding site to reduce binding affinity or shifting conformational equilibria to counteract the effects of antibiotic action. One of the major goals of our laboratory is to determine the structural basis of antibiotic resistance, information that can potentially accelerate the development of new antimicrobial agents effective against resistant pathogens. Our model system is the genetically tractable, extremely thermophilic bacterium Thermus thermophilus, whose ribosome structure has been determined at high resolution. We have identified a number of antibiotic-resistant mutants of T. thermophilus, and, through our ongoing collaboration with structural biologists, are establishing precise mechanisms of antibiotic resistance.
- The fidelity of mRNA decoding by the ribosome
Substrate selection by the ribosome is a highly accurate process that involves both local and large-scale conformational transitions. Interference with these transitions by certain antibiotics or by mutations in the ribosome can undermine translational fidelity. Aminoglycoside antibiotics, most notably streptomycin, reorganize the ribosome’s codon recognition active site into non-native conformations that impair its capacity to discriminate between correct and incorrect aminoacyl-tRNA substrates. While some mutations in the ribosome similarly promote miscoding, others are error-restrictive and cause the ribosome to translate more accurately than normal. Such mutations generally have significant fitness costs due to diminished protein synthesis kinetics. Experimental evolution allows us to identify secondary fitness-enhancing mutations, many of which are situated remote from the site of codon-recognition or the original mutation. Precisely how these mutations influence translational accuracy is not yet known, but understanding their nature is major goal of our laboratory as their wide dispersal suggests the existence of multiple long-range communication pathways throughout the ribosome during tRNA selection.
- Kamath D, Gregory ST, O’Connor M. 2017. The loop 2 region of ribosomal protein uS5 influences spectinomycin sensitivity, translational fidelity, and ribosome biogenesis. Antimicrobial Agents and Chemotherapy 61 (in press).
- VanNice J, Gregory ST, Kamath D, O’Connor M. 2016. Alterations in ribosomal protein L9 that decrease the fidelity of translation. Biochimie 128-129:122-126.
- Carr JF, Lee HJ, Jaspers JB, Dahlberg AE, Jogl G, Gregory ST. 2015. Phenotypic suppression of streptomycin resistance by mutations in multiple components of the translation apparatus. Journal of Bacteriology 197:2981–2988.
- Carr JF, Danziger ME, Huang AL, Dahlberg AE, Gregory ST. 2015. Engineering the genome of Thermus thermophilus using a counter-selectable marker. Journal of Bacteriology 197:1135-1144.
- Agarwal D, Kamath D, Gregory ST, O’Connor M. 2015. Modulation of decoding fidelity by ribosomal proteins S4 and S5. Journal of Bacteriology 197:1017-1025.
- Carr JF, Gregory ST, Dahlberg AE. 2015. Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus Extremophiles 19:221-228.
- Gregory ST, Connetti JL, Carr JF, Jogl G, Dahlberg AE. 2014. Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution. Journal of Bacteriology 196:3776–3783.
- Demirci H, Murphy FV, Murphy EL, Connetti JL, Dahlberg AE, Jogl G, Gregory ST. 2014. Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance. Antimicrobial Agents and Chemotherapy 58:4308–4317.
- Demirci H, Wang L, Murphy FV, Murphy EL, Carr JF, Blanchard SC, Jogl G, Dahlberg AE, Gregory ST. 2013. The central role of protein S12 in organizing the structure of the decoding site of the ribosome. RNA 19:1791–1801.
- Demirci H, Sierra RG, Laksmono H, Shoeman RL, Botha S, Barends TRM, Nass K, Schlichting I, Doak RB, Gati C, Williams GJ, Boutet S, Messerschmidt M, Jogl G, Dahlberg AE, Gregory ST, Bogan MJ. 2013. Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser Acta Crystallographica F69, 1066-1069.
- Demirci H, Murphy F, Murphy E, Gregory ST, Dahlberg AE, Jogl G. 2013. A structural basis for streptomycin-induced misreading of the genetic code. Nature Communications 4:1355-138.
- Agarwal D, Gregory ST, O’Connor M. 2011. Error-prone and error-restrictive mutations affecting ribosomal protein S12. Journal of Molecular Biology 410:1-9.
- O’Connor M, Gregory ST. 2011. Inactivation of the RluD pseudouridine synthase has minimal effects on growth and ribosome function in wild-type Escherichia coli and Salmonella enterica. Journal of Bacteriology 193:154-162.
- Demirci H, Murphy IV F, Belardinelli R, Kelley AC, Ramakrishnan V, Gregory ST, Dahlberg AE, Jogl G. 2010. Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function. RNA 16:2319-2324.
- Demirci H, Larsen LH, Hansen T, Rasmussen A, Cadambi A, Gregory ST, Kirpekar F, Jogl G. 2010. Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus. RNA 16:1584-1596.
- Gregory ST, Demirci H, Belardinelli R, Monshupanee T, Gualerzi C, Dahlberg AE, Jogl G. 2009. Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG. RNA 15:1693-1704.
- Demirci H, Belardinelli R, Seri E, Gregory ST, Gualerzi C, Dahlberg AE, Jogl G. 2009. Structural rearrangements in the active site of the Thermus thermophilus 16S rRNA methyltransferase KsgA in a binary complex with 5’-methylthioadenosine. Journal of Molecular Biology 388:271-282.
- Gregory ST, Dahlberg AE. 2009. Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA. RNA 15:215-223.
- Gregory ST, Carr JF, Dahlberg AE. 2009. A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA. RNA 15:208-214.
- Gregory ST, Dahlberg AE. 2008. Transposition of an insertion sequence, ISTth7, in the genome of the extreme thermophile Thermus thermophilus FEMS Microbiology Letters 289:187-192.
- Monshupanee T, Gregory ST, Douthwaite S, Chungjatupornchai W, Dahlberg AE. 2008. Mutations in the conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications. Journal of Bacteriology 190:7754-7761.
- Demirci H, Gregory ST, Dahlberg AE, Jogl G. 2008. Crystal structure of the Thermus thermophilus 16S rRNA methyltransferase RsmC in complex with cofactor and substrate guanosine. Journal of Biological Chemistry 283:26548-26556.
- Demirci H, Gregory ST, Dahlberg AE, Jogl G. 2008. Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase. Structure 16:1059-1066.
- Demirci H, Gregory ST, Dahlberg AE, Jogl G. 2007. Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA. EMBO Journal 26:567-577.
- Carr JF, Hamburg D-M, Gregory ST, Limbach PA, Dahlberg AE. 2006. Effects of streptomycin resistance mutations on posttranslational modification of ribosomal protein S12. Journal of Bacteriology 188:2020-2023.
- Suh M-J, Hamburg D-M, Gregory ST, Dahlberg AE, Limbach PA. 2005. Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry. Proteomics 5:4818-4831.
- Gregory ST, Carr JF, Rodriguez-Correa D, Dahlberg AE. 2005. Mutational analysis of 16S and 23S rRNA genes of Thermus thermophilus. Journal of Bacteriology 187:4804-4812.
- Carr JF, Gregory ST, Dahlberg AE. 2005. Severity of the streptomycin resistance and streptomycin dependence phenotypes of ribosomal protein S12 of Thermus thermophilus depends on the identity of highly conserved amino acid residues. Journal of Bacteriology 187:3548-3550.
- Gregory ST, Carr JF, Dahlberg AE. 2005. A mutation in the decoding center of Thermus thermophilus 16S rRNA suggests a novel mechanism of streptomycin resistance. Journal of Bacteriology 187:2200-2202.
- O’Connor M, Gregory ST, Dahlberg AE. 2004. Multiple defects in translation associated with altered ribosomal protein L4. Nucleic Acids Research 32:5750-5756.
- Cameron DM, Gregory ST, Thompson J, Dahlberg AE. 2004. Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly. Journal of Bacteriology 186:5819-5825.
- Cameron DM, Thompson J, Gregory ST, March PE, Dahlberg AE. 2004. Thiostrepton-resistant mutants of Thermus thermophilus. Nucleic Acids Research 32:3220-3227.
- Thompson J, Kim DF, O’Connor M, Lieberman KR, Bayfield MA, Gregory ST, Green R, Noller HF, Dahlberg AE. 2001. Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyl transferase active site of the 50S ribosomal subunit. Proceedings of the National Academy of Sciences of the USA 98:9002-9007.
- Gabashvili IS, Gregory ST, Valle M, Grassucci R, Worbs M, Wahl MC, Dahlberg AE, Frank J. 2001. The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Molecular Cell 8:181-188.
- O’ Connor M, Gregory ST, RajBhandary UT, Dahlberg AE. 2001. Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3. RNA 7:1-10.
- Gregory ST, Cate JH, Dahlberg AE. 2001. A spontaneous, erythromycin-resistance mutation in a 23S rRNA gene, rrlA, of the extreme thermophile Thermus thermophilus. IB-21. Journal of Bacteriology 183:4382-4385.
- Gregory ST, Cate JH, Dahlberg AE. 2001. Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus. Journal of Molecular Biology 309:333-338.
- Gregory ST, Dahlberg AE. 1999. Erythromycin resistance mutations in ribosomal proteins L4 and L22 perturb the higher order structure of 23S ribosomal RNA. Journal of Molecular Biology 289:827-834.
- Gregory ST, Dahlberg AE. 1999. Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23S ribosomal RNA. Journal of Molecular Biology 285:1475-1483.
- Gregory ST, O’Connor M, Dahlberg AE. 1996. Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium. Nucleic Acids Research 24:4918-4923.
- Gregory ST, Dahlberg AE. 1995. Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA. Nucleic Acids Research 23:4234-4238.
- Gregory ST, Dahlberg AE. 1995. Effects of mutations at position 36 of tRNAGlu on missense and nonsense suppression in Escherichia coli. FEBS Letters 361:25-28.
- Gregory ST, Lieberman KR, Dahlberg AE. 1994. Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy. Nucleic Acids Research 22:279-284.