B.A. Biology, Gustavus Adolphus College, 1992
We are interested in how the ribosome works. The ribosome is a large (~2.5 MDa), two-subunit, RNA-based machine that translates the genetic code in all organisms.
In recent years, numerous X-ray crystal structures of the ribosome and its isolated subunits and many cryo-EM reconstitutions of functional ribosomal complexes have been reported. Today, a primary challenge for the field is to elucidate the functional roles of specific structural elements of the ribosome. Since the ribosome is the most common target of natural antibiotics, gaining a better understanding of how the ribosome functions should contribute substantially to the development of new antibiotics. In our group, we use mutagenesis and antibiotics to address questions of ribosome structure and function. One of our primary interests is translocation, the coupled movement of tRNA and mRNA within the ribosome. In recent work, we have shown that destabilization of the codon-anticodon helix accompanies movement of tRNA from the P/P to P/E state, an important transition during translocation.
|McGarry, K. G., Walker, S. E., Wang, H. and Fredrick, K. 2005. Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state within the ribosome. Mol. Cell 20: 613-622.
Abdi, N. M. and Fredrick, K. 2005. Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in E. coli . RNA 11: 1624-1632.
Yassin, A., Fredrick, K. and A. S. Mankin. 2005. Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics. PNAS 102: 16620-16625.
Noller, H. F., Hoang, L. and Fredrick, K. 2005. The 30S ribosomal P site: A function of 16S rRNA. FEBS Letters 579: 855-858.
Hoang, L., Fredrick, K. and H. F. Noller. 2004. Creating ribosomes with an all-RNA 30S subunit P site. PNAS 101: 12439-12443.
Fredrick, K. and H. F. Noller. 2003. Catalysis of ribosomal translocation by sparsomycin. Science 300: 1159-1162.
Fredrick, K. and H. F. Noller. 2002. Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P site. Mol. Cell 9: 1125-1131.
Noller, H. F., Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Lancaster, L., Dallas, A., Fredrick, K., Earnest, T. N. and J. H. D. Cate. 2001. Structure of the ribosome at 5.5 � resolution and its interaction with functional ligands . Cold Spring Harbor Symp. Quant. Biol. 66: 57-66.
Fredrick, K., Dunny, G. M. and H. F. Noller. 2000. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30S subunits. J. Mol. Biol. 298: 379-394.
Immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis.
The leishmaniases comprise several diseases caused by intracellular protozoan parasites belonging to Leishmaniaspecies leading to a wide spectrum of clinical manifestations and a global health problem. Among the parasitic infections, this disease is responsible for the highest number of DALYs (Disability adjusted life years; a measure of health burden) after malaria. “Old world” cutaneous leishmaniasis usually manifests as a localized self-healing skin lesion with long-term protective immunity in humans. In contrast, some forms of “New world” cutaneous leishmaniasis manifests as a chronic infection that is associated with mutilation of ear and disfiguring scars or as a severe mucocutaneous disease involving nasal and oropharyngeal mucosa with extensive tissue destruction. Visceral leishmaniasis is the most severe clinical form, characterized by hepatosplenomegaly, fever, abdominal pain and weight loss.
Our laboratory is interested in understanding the immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis caused by L. mexicana and L. donovani respectively. We are particularly interested in studying the role of cytokines in regulation of immune responses during these two species of Leishmaniaand the use of cytokine and cytokine receptor gene deficient mice has been a very powerful tool in these studies. As cytokines can modulate functions of several cells of the immune system in vivo, we are now using cell-specific gene deficient mice lacking specific cytokine receptors on specific immune cells such as macrophages and T cells. These mice are generated using cre/lox technology that enables us to delete a gene in cell-specific manner. We believe that these studies will enable us to determine how cytokines regulate immune responses in vivo during leishmaniasis. With regards to L. donovani, our studies have focused on understanding the regulation of effector cell responses in murine visceral leishmaniasis caused by L. donovani. Of particular interest to our group is the determining the immune mechanisms that mediate protection and/or induce immunopathology during VL. More recently, in collaboration with the McGill University, we have initiated studies that focus on the development of amastigote-specific single candidate vaccine against visceral and “New world” cutaneous leishmaniasis that cause considerable morbidity and mortality in humans. Another area of research in our laboratory is understanding the immunological basis of gender-related differences in susceptibility to Leishmania. In these studies, we are interested in determining the roles of sex-hormones in modulation of immune response and determining the outcome of Leishmania infection. Our long-term goal is to identify the basic mechanisms by which cytokines regulate T cell responses and host immunity to cutaneous leishmaniasis caused by L. mexicana and visceral leishmaniasis caused by L. donovani and utilize this knowledge to develop a vaccine against these diseases.
Xu D, McSorley SJ, Tetley L, Chatfield S, Dougan G, Chan WL, Satoskar, AR, David JR, Liew FY. Protective effect on Leishmania major infection of MIF, TNF-alpha and IFN-gamma administered orally via attenuated Salmonella typhimurium. J. Immunol. 1998; 160: 1285-1289.
Satoskar AR, Khamis Al-Q, Alexander J. Sex-determined resistance against Leishmania mexicana is associated with the preferential induction of a Th1-like response and IFN-gamma production by female but not male DBA/2 mice. Immun Cell Biol. 1998; 76: 159-166.
Satoskar, AR, Okano M, Connaughton S, David JR, Labow M. Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice. Europ J Immunol.1998; 28: 2066-2074.
Stamm L, Raisanen-Solokowski A, Okano M, Russell M, David JR, Satoskar AR. Mice with STAT6-targeted disruption develop a Th1 response and control cutaneous leishmaniasis. J. Immunol. 1998; 161: 6180-6188.
Bozza M, Soares M, Bozza P, Satoskar AR, Brombacher F, Titus R, Shoemaker C, David JR. The selective PACAP-type I receptor agonist Maxadilan from Sand fly saliva protects mice against lethal endotoxemia. Europ J Immunol. 1998; 28: 3120-3127.
Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J. Exp. Med. 1999; 189: 341-346.
Satoskar AR, Stamm LM, Zhang, XM, Satoskar, AA, Okano, M, David JR, Terhorst C, Wang B. Mice lacking natural killer (NK) cells develop an efficient Th1 response and control cutaneous L. major infection. J. Immunol. 1999; 162:6747-6754.
Satoskar, AR, Stamm, LM, Zhang, X., Okano, M, David, JR, Terhorst, C., and Wang, B. NK cell deficient mice develop Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response. J. Immunol. 1999; 163: 5298-5302.
Stamm, LM, Satoskar, AA, Ghosh, S, David, JR and Satoskar, AR. STAT4-mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis. Europ. J. Immunol. 1999; 29:2524-2529.
Alexander, J, Satoskar AR and Russell, DG. Leishmania species: models of intracellular parasitism. J. Cell Sci.1999; 112:2993-3002. (Review).
Satoskar, AR, Rodig, S, Telford, SR, Satoskar, AA, Ghosh, S., von Lichtenberg, F., and David, JR. Interleukin 12 gene deficient mice are susceptible to L. donovani infection but have diminished hepatic immunopathology. Europ. J. Immunol. 2000; 30:834-839..
Monteforte, G., Takeda, K., Akira, S, David, JR and Satoskar, AR. Interleukin-18 is not critical for the development of Th1 response and control of cutaneous L. major infection. J. Immunol. 2000; 164: 5890-5893.
Alexander, J., Carter, KC., Al-Fasi, N., Satoskar, AR, and Brombacher, F. Effective drug therapy against visceral leishmaniasis is dependent on endogenous IL-4. Europ. J. Immunol. 2000; 30:2935-2943.
Hattori, H, Okano, M, Yoshino, T, Akagi, T, Nakayama, E, Saito, C, Satoskar, AR, Ogawa, T, Azuma, M and Nishizaki, K. Expression of co-stimulatory CD80/CD86-CD28/CD152 molecules in the nasal mucosa of patients with perennial allergic rhinitis. Clin. Exp. Allergy. 2001; 31:1242.
Okano, M, Azuma, M, Yoshino, T, Hattori, H, Nakada, M, Satoskar, AR, Harn Jr, DA, Nakayama, E, Akagi, T, and Nishizaki, K. Differential role of CD80 and CD86 molecules in the induction and the effector phases of allergic rhinitis in mice. Am. J. Res. Crtic. Care Med. 2001; 164: 1501.
Szabo, SJ, Sullivan, BM, Stemmann, C., Satoskar, AR, Sleckman, BP, and Glimcher, LH. Distinct effects of T-bet in Th1 lineage commitment and IFN-γ production in CD4 and CD8 T cells. Science. 2002; 295:338.
Greenwald, RJ, McAdam, AJ, Van der Woude, D, Satoskar, AR, and Sharpe, AH. Inducible co-stimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis. J. Immunol. 2002; 168: 991.
Rodriguez-Sosa, M, David, JR, Bojalil, R., Satoskar, AR, and Terrazas, LI. Susceptibility to the larval stage of the helminth parasite Taenia crassiceps is mediated by the Th2 response induced via STAT6 signaling. J. Immunol. (Cutting Edge) 2002; 168:3135.
Brown, JA, Greenwald, RJ, Scott, S, Schweitzer, A.N, Satoskar, AR, Chung, C, Schopf, LR, van der Woude, D, Sypek, JP, and Sharpe, AH. T helper differentiation in resistant and susceptible B7-deficient mice infected with Leishmania major. Eur. J. Immunol. 2002; 32:1764.
Hattori H, Okano M, Yamamoto T, Yoshino T, Yamashita Y, Watanabe T, Satoskar, AR, Harn DA, and Nishizaki K. Intranasal application of purified protein derivative suppresses the initiation but not the exacerbation of allergic rhinitis in mice. Clin. Exp. Allergy. 2002; 32:951.
Costa, CH, Stewart, JM, Gomes, RBB, Garcez, LM, Ramos, PK, Bozza, M, Satoskar, AR, Dissennayake, S, Santos, RS, Silva, MRB, Shaw, JJ, David, JR, and Maguire, JH. Asymptomatic human carriers of Leishmania chagasi. Am. J. Trop. Med. Hyg. 2002; 66:334-337.
Rodriguez-Sosa, M, Satoskar, AR, Calderon, R., Gomez-Garcia, L, Saaverda, R., Bojalil, R., and Terrazas, LI. Chronic helminth infection induces alternatively activated macrophages expressing high levels of CCR5 with low interleukin-12 production and Th2-biasing ability. Infect. Immun. 2002; 70:3656.
Parish, CL, Finkelstein, DI, Tripanichkul, W, Satoskar, AR, Drago, J, and Horn, MK. The role of Interleukin-1, Interleukin-6 and glia in inducing growth of neuronal terminal arbors in mice. J. Neurosci. 2002; 22:8034.
Wurster, AL, Rodgers, VL, Satoskar, AR., Whitters, MJ, Young, DA, Collins, M, and Grusby, MJ. Interleukin-21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naïve Th cells into interferon gamma-producing Th1 cells. J. Exp. Med. 2002; 196: 969.
Pien, GC, Nguyen, KB, Malmgaard, L, Satoskar, AR, and Biron, CA. A unique mechanism for innate cytokine promotion of T cell responses to viral infections. J. Immunol. 2002; 169:5827.
Rodriguez-Sosa, M, Rosas, LE, David, JR, Bojalil, R, Satoskar, AR*, and Terrazas, LI*. Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps. (*Joint senior co-authors) Infect. Immun. 2003; 71:1247.
Rodriguez-Sosa, M, Rosas, LE, Terrazas, LI, Lu, B, Gerard, C, and Satoskar, AR*. CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection. Immunol. Cell Biol. 2003; 80:114.
Rodriguez-Sosa, M, Satoskar, AR, David, JR, and Terrazas, LI. Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps. Int. J. Parasitol. 2003; 33: 701.
Rosas, L, Keiser, T, Pyles, R, Durbin, J, and Satoskar, AR*. Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway. Eur. J. Immunol. 2003; 33: 1799.
Pan, J.H., Sukhova, G.K., Satoskar, A.R., David, J.R., Yang, J.T., Fu, H., Metz, C., Baugh, J.A., Bucala, R., Fang, K., Libby, P. and Shi, G.P. Regulation of cysteine protease expression by macrophage migration inhibitory factor. Circulation. 2004; 109:3149-3153.
Rodriguez-Sosa, M., Rosas, L.E., Saavedra, R., Satoskar, A.R. and Terrazas, L.I. STAT4-dependent IL-12 signaling pathway is required for resistance to the helminth parasite Taenia crassiceps. Infect. Immun. 2004; 71:1247-1254.
Wang, N., Satoskar, A.R., Faubion, W., Howie, D., Okamoto. S., Feske, S., Gullo, C., Clarke, K., Rodriguez Sosa, M., Sharpe, A.H. and Terhorst, C. SLAM controls T cell and macrophage functions. J. Exp. Med. 2004; 199:1255-1264.
Bhardwaj, N., Rosas, L.E., Lafuse, W.P., and Satoskar, A.R. Leishmania inhibits STAT1-mediated IFN-γ signaling in macrophages: Increased tyrosine phosphorylation of dominant negative STAT1β by Leishmania mexicana. Int. J. Parasitol. 2005; 35:7582.
Howie, D., Laroux, F.S., Morra, M., Satoskar, A.R., Rosas, L.E., Faubion, W.A., Julien, A., Rietdijk, S., Coyle, A.J., Fraser, C., and Terhorst, C. The SLAM family receptor Ly108 controls T cell and neutrophil functions. J. Immunol. 2005; 174:5931-5935.
Morra, M., Barrington, R.A., Abadia-Molina, A., Okamoto, S., Julien, A., Gullo, C., Kalsy, A., Edwards, M.J., Chen, G., Spolski, R., Leonard, W.J., Huber, B.T., Borrow, P., Biron, C.A., Satoskar, A.R., Carroll, M.C., and Terhorst, C. Defective B cell responses in the absence of SH2D1A. Proc. Natl Acad. Sci. USA. 2005; 102:4819-4823.
Okano, M., Hattori, H., Yoshino, T., Sugata, Y., Yamamoto, M., Fujiwara, T., Satoskar, A.A., Satoskar, A.R., and Nishizaki, K.Nasal exposure to Staphylococcal enterotoxin enhances the development of allergic rhinitis in mice. Clin. Exp. Allergy. 2005; 35:506-514.
Powell, N.D., Papenfuss, T.L., McClain, M.A., Gienapp, I.E., Shawler, T.M., Satoskar, A.R., and Whitacre, C.C. Macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis. J. Immunol. 2005; 175:5611-5614.
Rosas, L.E., Barbi, J., Lu, B., Fujiwara, N., Gerard, C., Sanders, V.M., and Satoskar A.R. CXCR3-/- mice mount an efficient Th1 response but fail to control L. major infection. Eur. J. Immunol. 2005; 35:515-523.
Rosas, L.E., Keiser, T., Barbi, J., Satoskar, A.A., Septer, A., Kcazmarek, J., Lezama-Davila, C.M., and Satoskar, A.R. Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice. Int. Immunol. 2005; 17:1347-1357.
Hattori, H., Okano, M., Kariya, S., Nishizaki, K. and Satoskar, A. R. CD40-CD40L interaction is involved in pathogenesis of SEA induced allergic rhinitis. Amer. J. Rhinol. 2006; 20:165-169.
Liang, S.C., Greenwald, R.J., Latchman, Y.E., Rosas, L., Satoskar, A. R, Freeman, G.J. and Sharpe, A.H. PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis. Eur. J. Immunol. 2006; 36:58-64.
Reyes, J., Terrazas, L.I., Espinoza, B., Gomez-Garcia, L., Cruz-Robles, D., Rivera-Montoya, I., Snider, H., Satoskar, A. R. and Rodriguez-Sosa, M. Macrophage migration inhibitory factor (MIF) plays a critical role in host defense against acute Trypanosoma cruzi infection. Infect. Immun. 2006; 74:3170-3179.
Rosas, L.E., Barbi, J., Snider, H., Satoskar, A.A., Lugo-Villarino, G., Keiser,T., Papenfuss, T, Durbin, J, Radzioch, D, Glimcher, LH and Satoskar, A. R. Cutting edge: STAT1 and T-bet play distinct roles in determining outcome of visceral leishmaniasis caused by Leishmania donovani J. Immunol. 2006; 177:22-25.
Rosas, L.E., Satoskar, A.A., Roth, K., Keiser, T., Barbi, J., Hunter, C.A, de Sauvage, F. and Satoskar, A. R. IL-27R (WSX-1/TCCR) gene deficient mice display enhanced resistance to Leishmania donovani infection but develop severe liver immunopathology. Am. J. Pathol. 2006; 168:158-169.
|Tina M. Henkin
B.A., Biology, Swarthmore College
Professor of Microbiology
Robert W. and Estelle S. Bingham Professor of Biological
Member, Center for RNA Biology
Member, OSBPRiboswitch RNAs; Transcription termination; Translation initiation; RNA structure/function; antibiotic design; Gram-positive bacteria
The main area of interest in our laboratory is the analysis of the mechanisms through which cells sense changes in their environment and transmit that information to the level of gene expression. We use the Gram-positive bacterium Bacillus subtilis as a model system, and we focus primarily on genes involved in protein synthesis and amino acid metabolism. We have uncovered systems in which nascent RNA transcripts act as riboswitches to directly sense physiological signals and control gene expression through RNA structural rearrangements.
Nascent RNAs can sense uncharged tRNA: the T box system
Characterization of the B. subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, revealed a novel mechanism of gene regulation at the level of transcription antitermination. The tyrS gene is a member of a large family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria that are regulated by a common mechanism. Each gene in this family responds individually to limitation for the appropriate amino acid. Amino acid limitation is monitored via interaction of the 5’ region of the nascent transcript with the cognate uncharged tRNA. This interaction is directed by pairing of the anticodon of the tRNA with a single codon, designated the “Specifier Sequence,” in the mRNA. The mRNA-tRNA interaction occurs in the absence of translation, and antitermination can occur in a purified transcription system with no additional cellular factors, indicating that the mRNA is sufficient for specific recognition of the cognate tRNA. We are currently investigating the molecular details of the leader RNA-tRNA interaction, and the structural shifts in both RNA partners that occur upon binding. We are also testing novel antibiotics for their ability to target the T box mechanism.
Nascent RNAs can sense small molecules: metabolite-binding riboswitch RNAs
Analysis of genes involved in methionine metabolism revealed a second global transcription antitermination system, dedicated to genes in this pathway. Like the T box system, the S box system is widely used in Gram-positive organisms. Genes regulated by this mechanism contain highly conserved sequence and structural elements in their mRNAs, and expression is induced by starvation for methionine. We have now shown that the molecular effector for this system is S -adenosylmethionine, which binds directly to the leader RNA and modululates its structure to promote transcription termination. A second SAM-binding RNA, the SMK box, was identified in lactic acid bacteria and shown to regulate gene expression at the level of translation initiation. We have also shown that lysine biosynthesis genes are regulated by a similar mechanism, with specific leader RNA binding of lysine. Current work is focusing on the molecular mechanisms of effector recognition and RNA rearrangement in response to effector binding.
Molecular mechanisms of transcription and transcriptional regulation, including elongation control of virulence genes in proteobacteria.
The focal point of the research in our lab is RNA polymerase (RNAP), the enzyme that is responsible for the first step in gene expression, mRNA synthesis. RNAP accomplishes this task during the transcription cycle that is composed of three major steps: initiation, elongation, and termination. All these steps are subject to elaborate control by numerous regulatory proteins and small effectors. RNAP is also an attractive target for antibacterial drugs. Using a combination of biochemical, genetic, and structural (in collaboration with Dr. Vassylyev’s Lab at UAB) approaches, we are currently working on several projects:
Substrate selection by RNAP
To transmit genetic information from genome to proteome in undistorted form, RNAP must synthesize the nascent RNA with high fidelity. Fidelity mechanisms are well-studied in DNA polymerases, and to a lesser extent in single-subunit (phage T7) RNAPs . However, the mechanism of substrate selection by multi-subunit enzymes has not been studied. We are utilizing structure-based mutagenesis to elucidate this mechanism of correct nucleotide selection, and have already obtained a set of RNAP variants with altered substrate selection properties.
Mechanism and regulation of RNA chain elongation and termination
Tth RNAP elongation complex
The rate of transcription is determined by the nucleic acid signals that slow transcription. These signals serve as regulatory checkpoints at which RNAP could be modified by action of auxiliary factors, and therefore determine the gene expression patterns in all organisms. We want to determine how certain DNA and RNA sequences trigger RNAP isomerization into an un-reactive, slow state, which is characterized by a dramatic decrease in the rate of nucleotide addition, and is a likely target for elongation factors (such as NusA, NusG and RfaH). We study how RNAP itself recognizes transcription roadblocks and how auxiliary factors affect its behavior.
RfaH, an elongation enhancer protein
Efficient synthesis of long messages relies on transcription factors that allow RNAP to overcome transcription roadblocks. RfaH is a bacterial antitermination factor that enables RNAP to transcribe through long polycistronic operons encoding toxins, antibiotics, capsules, lipopolysaccharide core, and F-pili, all of which are molecules that contribute to bacterial pathogenesis. We are studying the molecular mechanism by which RfaH “switches” RNAP into a highly processive state, pursuing determination of the X-ray structure of RfaH, characterizing the RfaH regulon in E. coli , and conducting the comparative analysis of RfaH orthologs from different bacteria (Y. enterocolitica, V. cholerae, K. pneumoniae, etc.).
Molecular mechanisms of transcriptional inhibitors action on RNAP
Rifabutin and rifapentin bound to Tth RNAP holoenzyme, solution.
Inhibitors of bacterial RNAP are used as antibiotics to treat bacterial infections and in research to gain insights into molecular mechanisms that regulate transcription. We are working on the mechanism of RNAP inhibition by rifamycins, tagetitoxin, and CBRs. We perform detailed analysis of the mechanism of action of these inhibitors by a combination of genetic and biochemical techniques and collaborate with Dr. Vassylyev to obtain high-resolution X-ray structures of the Thermus thermophilus RNAP in complex with these drugs. We plan to use the collected data for design ofnovel antibiotics.
Regulation of transcription through RNAP secondary channel
RNAP secondary channel postulated to facilitate delivery of substrate NTPs to the active site appears to facilitate access of other small molecules and auxiliary factors to the catalytic center of the enzyme. Alarmone ppGpp, inhibitor of chloroplast development tagetitoxin, cleavage factors GreA and GreB, and their structural analog DksA all have been recently added to the growing list of transcriptional regulators utilizing secondary channel as the only accessible venue connecting RNAP active site to the surface of the enzyme. Presently we collaborate with Dr. Vassylyev’s Laboratory at UAB in elucidating the mechanisms by which these and other factors regulate activity of the RNA polymerase.