Paul Babitzke obtained his B.A. in Biomedical Science at St. Cloud State University and his Ph.D. in Genetics from the University of Georgia. He worked as a postdoctoral scientist for 3 years at Stanford University prior to joining the faculty at Penn State University in 1994. He is currently Professor of Biochemistry and Molecular Biology and Director of the Center for RNA Molecular Biology.
Regulation of gene expression by RNA structure and RNA-binding proteins
Gene expression is regulated at the level of transcription, translation and mRNA stability. The transcription cycle consists of initiation, elongation and termination, each of which can be regulated. We are investigating fundamental mechanisms that affect transcription elongation and termination. We also investigate a variety of genes in which RNA binding proteins control gene expression by transcription attenuation (regulated termination), repression of translation initiation, and/or mRNA stability.
The Bacillus subtilis trp operon is regulated by TRAP-mediated transcription attenuation and translation repression mechanisms. The general transcription elongation factors NusA and NusG participate in the attenuation mechanism by stimulating RNA polymerase pausing at the nucleotide just preceding the critical overlap between the antiterminator and terminator structures. Thus, NusA- and NusG-dependent pausing provides additional time for TRAP to bind to the nascent transcript and promote termination. TRAP also regulates translation of the trp operon. TRAP binding to trp operon readthrough transcripts promotes formation of an RNA structure that prevents ribosome binding. NusA- and NusG-dependent pausing plays a vital role in this mechanism as well. NusA stimulates pausing by assisting with pause hairpin formation, whereas NusG makes sequence-specific contacts with T residues in the non-template DNA (ntDNA) strand within the paused transcription bubble. As RNAP and template DNA must move with respect to one another for elongation to resume, interaction of NusG with both components inhibits elongation.
Until recently the two trp pause sites were the only two known NusG-dependent pause sites in any organism. Using a genomic approach called RNET-seq, we identified >1000 NusG-dependent pause sites in the B. subtilis transcriptome. Pausing at one such site is required for inducing expression of tlrB, a gene encoding a 23S rRNA methyltransferase. TlrB-mediated methylation confers tylosin resistance by preventing binding of this antibiotic to the ribosome. Pausing is also required for regulating expression of the ribD riboswitch by providing additional time for FMN binding to the nascent ribD transcript. We are also exploring the mechanism by which NusA- and NusG-dependent pausing controls expression of other genes in B. subtilis.
Intrinsic (Rho-independent) and Rho-dependent termination are the two transcription termination mechanisms identified in bacteria. Canonical intrinsic terminators consist of a strong RNA hairpin followed by a U-tract. Term-seq studies identified a new class of intrinsic terminator that requires NusA. NusA-dependent terminators tend to have weak RNA hairpins and/or distal U-tract interruptions. Our unpublished studies indicate that NusG is another intrinsic termination factor. We are exploring the interrelationship between NusG-dependent pausing and NusG-dependent termination. Our hypothesis is that NusG stimulates pausing prior to transcript release at NusG-dependent termination sites. We are conducting biochemical studies to test this hypothesis. We are also exploring the effects of Rho on termination. Of particular interest, we found that Rho actually participates in intrinsic (Rho-independent) termination. We are exploring the mechanistic basis for this important discovery.
We are also using RNA structure-seq to identify new regulatory mechanisms (attenuation, antitermination, riboswitches, RNA thermometers) that control gene expression in response to various stresses. This work is being conducted in collaboration with Drs. Philip Bevilacqua and Sarah Assmann at Penn State.
Another major effort in the lab involves genetic and biochemical characterization of the Csr global regulatory system. Csr regulates several hundred E. coli genes, thereby mediating global changes in cellular physiology during the transition between exponential and stationary phase growth. Four major components of Csr include an RNA binding protein (CsrA), two small RNA (sRNA) antagonists of CsrA (CsrB and CsrC), and CsrD, a protein that targets degradation of CsrB and CsrC by RNase E.
CsrA represses translation initiation of numerous genes by binding to their translation initiation regions. Bound CsrA prevents ribosome binding, thereby repressing a variety of processes, including gluconeogenesis, glycogen biosynthesis, quorum sensing and biofilm formation. In contrast, CsrA activates glycolysis, acetate and iron metabolism, and motility. CsrA activates flagella biosynthesis by preventing degradation of the flhDC transcript by RNase E. We recently identified the first known CsrA-mediated translational activation mechanism. We are continuing to explore this global regulatory system in E. coli using omics, genetics and biochemistry approaches. For example, we are using structure-seq to footprint CsrA across the entire transcriptome in vivo. We have also initiated a metabolomics study to identify new pathways that are affected by the Csr system. All of the Csr work is being performed in collaboration with Dr. Tony Romeo at the University of Florida.
Honors and Awards
- Chair, NIGMS Microbial Physiology and Genetics-subcommittee 2 (MBC-2) (2004)
- Chair, Division H, American Society for Microbiology (ASM) (2006)
- Daniel R. Tershak Teaching Award (2009)
- Divisional Group IV Representative, American Society for Microbiology (ASM) (2011-2015)
- Charles E. Kaufman New Initiative Research Award (2016)
- Fellow, American Association for the Advancement of Science (AAAS) (2016)
- Fellow, American Academy of Microbiology (AAM) (2017)
- St. Cloud State University Biological Sciences Distinguished Alumni Award (2018)
- Babitzke, P., and Kushner, S.R. (1991) The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli. Proc. Natl. Acad. Sci. USA. 88:1-5.
- Babitzke, P., and Yanofsky, C. (1993) Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein. Proc. Natl. Acad. Sci. USA. 90:133-137.
- Babitzke, P., Bear, D.G., and Yanofsky, C. (1995) TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides. Proc. Natl. Acad. Sci. USA. 92:7916-7920.
- Du, H., Tarpey, R., and Babitzke, P. (1997) The trp RNA-binding attenuation protein regulates TrpG synthesis by binding to the trpG ribosome binding site of Bacillus subtilis. J. Bacteriol. 179:2582-2586.
- Du, H., and Babitzke, P. (1998) trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis. J. Biol. Chem. 273:20494-20503.
- Baker, C.S., Morozov, I., Suzuki, K., Romeo, T., and Babitzke, P. (2002) CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli. Mol. Microbiol. 44:1599-1610.
- Yakhnin, A.V., and Babitzke, P. (2002) NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro. Proc. Natl. Acad. Sci. USA. 99:11067-11072.
- Deikus, G., Babitzke, P., and Bechhofer, D.H. (2004) Recycling of an RNA-binding protein by ribonuclease digestion. Proc. Natl. Acad. Sci. USA 101:2747-2751.
- Gollnick, P., Babitzke, P., Antson, A., and Yanofsky, C. (2005) Complexity in regulation of tryptophan biosynthesis in Bacillus subtilis. Ann. Rev. Genet. 39:47-68.
- Dubey, A.K., Baker, C.S., Romeo, T., and Babitzke, P. (2005) RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction. RNA 11:1579-1587.
- Wang, X., Dubey, A.K. Suzuki, K., Baker, C.S., Babitzke, P., and Romeo, T. (2005) CsrA post-transcriptionally represses pgaABCD, responsible for the synthesis of a biofilm polysaccharide adhesin of Escherichia coli. Mol. Microbiol. 56:1648-1663.
- Suzuki, K., Babitzke, P., Kushner, S.R., and Romeo, T. (2006) Identification of a novel regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E. Genes Dev. 20:2605-2617.
- Yakhnin, A.V., Yakhnin, H., and Babitzke, P. (2006) RNA polymerase pausing participates in the Bacillus subtilis trpE translation control mechanism by providing additional time for TRAP to bind to the nascent trp leader transcript. Mol. Cell 24:547-557.
- Yakhnin, H., Pandit, P., Petty, T.J., Baker, C.S., Romeo, T., and Babitzke, P. (2007) CsrA of Bacillus subtilis regulates translation initiation of the gene encoding the flagellin protein (hag) by blocking ribosome binding. Mol. Microbiol. 64:1605-1620.
- Baker, C.S., Eöry, L.A., Yakhnin, H., Mercante, J., Romeo, T., and Babitzke, P. (2007). CsrA inhibits translation initiation of Escherichia coli hfq by binding to a single site overlapping the Shine-Dalgarno sequence. J. Bacteriol. 189:5472-5481.
- Yakhnin, A.V., Yakhnin, H., and Babitzke, P. (2008) Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader. Proc. Natl. Acad. Sci. USA 105:16131-16136.
- Babitzke, P., Baker, C.S., and Romeo, T. (2009) Regulation of translation initiation by RNA binding proteins. Ann. Rev. Microbiol. 63:27-44.
- McGraw, A.P., Mokdad, A., Major, F., Bevilacqua, P.C., and Babitzke, P. (2009) Molecular basis of TRAP-5’SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism. RNA 15:55-66.
- Yakhnin, A.V., and Babitzke, P. (2010) Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader. Mol. Microbiol. 76:690-705.
- Edwards, A.N., Patterson-Fortin, L.M., Vakulskas, C.A., Mercante, J.W., Potrykus, K., Vinella, D., Camacho, M.I., Fields, J.A., Thompson, S.A., Georgellis, D., Cashel, M., Babitzke, P., and Romeo, T. (2011) Circuitry linking the Csr and stringent response global regulatory systems. Mol. Microbiol. 80:1561-1580.
- Yakhnin, H., Yakhnin, A.V., Baker, C.S., Sineva, E., Berezin, I., Romeo, T., and Babitzke, P. (2011) Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by E70 and ES, and indirect transcriptional activation by CsrA. Mol. Microbiol. 81:689-704.
- Mukherjee, S., Yakhnin, H., Kysela, D., Sokoloski, J., Babitzke, P., and Kearns, D.B. (2011) CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis. Mol. Microbiol. 82:447-461.
- Yakhnin, A.V., Baker, C.S., Vakulskas, C.A., Yakhnin, H., Berezin, I., Romeo, T., and Babitzke, P. (2013) CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage. Mol. Microbiol. 87:851-866.
- Yakhnin, A.V., and Babitzke, P. (2014) NusG/Spt5: are there universal functions of this ubiquitously conserved transcription elongation factor? Curr. Opin. Microbiol. 16:68-71.
- Vakulskas, C.A., Potts, A.H., Babitzke, P., Ahmer, B.M., and Romeo, T. (2015) Regulation of bacterial virulence by Csr (Rsm) systems. Microbiol. Mol. Biol. Rev. 79:193-224.
- Yakhnin, H., Yakhnin, A.V., and Babitzke, P. (2015) Ribosomal protein L10(L12)4 autoregulates expression of the Bacillus subtilis rplJL operon by a transcription attenuation mechanism. Nucleic Acids Res. 43:7032-7043.
- Park, H., Yakhnin, H., Connolly, M., Romeo, T., and Babitzke, P. (2015) CsrA participates in a PNPase autoregulatory mechanism by selectively repressing translation of pnp transcripts that have been previously processed by RNase III and PNPase. J. Bacteriol. 197:3751-3759.
- Mondal, S., Yakhnin, A.V., Sebastian, A., Albert, I., and Babitzke, P. (2016) NusA-dependent transcription termination prevents misregulation of global gene expression. Nat. Microbiol. 1:15007.
- Yakhnin, A.V., Murakami, K.S., and Babitzke, P. (2016) NusG is a sequence-specific RNA polymerase pause factor that binds to the non-template DNA within the paused transcription bubble. J. Biol. Chem. 291:5299-5308.
- Pannuri, A., Vakulskas, C.A., Zere, T., McGibbon, L.C., Edwards, A., Ahmer, B., Babitzke, P., and Romeo, T. (2016) Circuitry linking the catabolite repression and Csr global regulatory systems of Escherichia coli. J. Bacteriol. 198:3000-3015.
- Mukherjee, S., Oshiro, R., Yakhnin, H., Babitzke, P., and Kearns, D.B. (2016) FliW antagonizes CsrA by a novel non-competitive allosteric mechanism. Proc. Natl. Acad. Sci. USA 113:9870-9875.
- Park,H., McGibbon, L.C., Potts, A.H., Yakhnin, H., Romeo, T., and Babitzke, P. (2017) Translational repression of the RpoS antiadapter IraD by CsrA is mediated via translational coupling to a short open reading frame. mBio 8:e01355-17.
- Yakhnin, H., Aichele, R., Ades, S.E., Romeo, T., and Babitzke, P. (2017) Circuitry linking the global Csr and E-dependent cell envelope stress response systems. J. Bacteriol. 199:e00484-17.
- Potts, A.H., Vakulskas, C.A., Pannuri, A., Yakhnin, H., Babitzke, P., and Romeo, T. (2017) Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics. Nat. Commun. 8:1596.
- Mitchell, D. III, Renda, A.J., Douds, C.A., Babitzke, P., Assmann, S.A., and Bevilacqua, P.C. (2019) In vivo RNA structural probing of uracil and guanine base pairing by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). RNA 25:147-157.
- Pourciau, C., Pannuri, A., Potts, A., Yakhnin, H., Babitzke, P., and Romeo, T. (2019) Regulation of iron storage by CsrA supports exponential growth of Escherichia coli. mBio 10:e01034-19.
- Babitzke, P., Lai, Y-J, Renda, A.J., and Romeo T. (2019) Post-transcription initiation control mediated by bacterial RNA binding proteins. Ann. Rev. Microbiol. 73:43-67.
- Yakhnin, H., Yakhnin, A.V., Mouery, B., Mandell, Z.F., Karbasiafshar, C., Kashlev, M., and Babitzke, P. (2019). NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling lead to antibiotic resistance by inducing 23S rRNA methylation. mBio:e02665-19.