Biochemistry and Molecular Biology
Penn State Science
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Paul Babitzke

Paul Babitzke

Main Content

  • Professor of Biochemistry and Molecular Biology
  • Graduate Education Co-Director
  • Co-director, Center for RNA Molecular Biology
203 Althouse Laboratory
University Park, PA 16802
Email: pxb28@psu.edu
Phone: (814) 865-0002

Research Interests

Regulation of gene expression by RNA structure and RNA-binding proteins

Graduate Programs

BMMB

Research Summary

Gene expression can be 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 are also investigating 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. When activated by tryptophan, TRAP binds to the nascent trp transcript and prevents formation of an antiterminator structure, thereby allowing formation of an overlapping transcription terminator, which halts transcription before RNA polymerase reaches the trp structural genes. In the absence of tryptophan TRAP does not bind to the RNA such that formation of the antiterminator allows transcription into the structural genes. 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-stimulated pausing provides additional time for TRAP to bind to the 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- stimulated pausing plays a vital role in this mechanism as well. In addition to exploring these pausing mechanisms, we are using genomic approaches (NET-seq, RNET-seq) to identify additional pause sites that are stimulated by NusA and/or NusG.

Canonical intrinsic transcription terminators consist of a contiguous RNA hairpin followed by a U-rich tract. We recently demonstrated that NusA is required for termination at non-canonical terminators that have interrupted hairpins and/or those with a minimal U-rich tract. Thus, NusA-dependent termination constitutes a previously unrecognized transcription termination mechanism in bacteria. Of particular interest, we identified a novel transcription attenuation mechanism in which NusA autoregulates its expression via NusA-dependent termination. We are now using RNA-seq to identify genomic termination defects caused by the loss of NusA, NusG and/or Rho.

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. Phil 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 ~700 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 metabolism and flagella biosynthesis. CsrA activates flagella biosynthesis by preventing degradation of the flhDC transcript by RNase E. We continue to explore this global regulatory system in E. coli and B. subtilis from biochemical and genomic viewpoints.

Babitzke figure 1

Model of the termination/pausing decision in the B. subtilis trp leader
(a) RNAP with bound NusA and NusG arrives at the termination site. In the presence of bound TRAP the antiterminator does not form. Thus, NusA-stimulated folding of the terminator/pause hairpin leads to termination (not shown). In the absence of bound TRAP the antiterminator forms, thereby preventing completion of the terminator/pause hairpin. Thus, RNAP transcribes past the terminator.
(b) RNAP with bound NusA and NusG arrives at the U144 pause site and RNAP enters into an elemental pause state. NusA-stimulated completion of the terminator/pause hairpin via branch migration stabilizes the pause. Sequence-specific interaction of NusG with T137-T139 of the non-template DNA strand further stabilizes the pause at U144.

 

Babitzke figure 2

Model of CsrA-RNA interaction
(a) Each CsrA dimer contains two symmetric RNA binding surfaces. CsrA initially interacts with a high-affinity site within the loop of a short RNA hairpin.
(b) The initial interaction increases the localized concentration of CsrA such that it can bind to a low-affinity site overlapping the target mRNA's Shine-Dalgarno (SD) sequence.
(c) Bound CsrA blocks ribosome binding, thereby repressing translation initiation.

Selected Publications

  • Yakhnin, Alexander V., and Paul Babitzke. (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, Gintaras, Paul Babitzke, and David H. Bechhofer. (2004) Recycling of an RNA-binding protein by ribonuclease digestion. Proc. Natl. Acad. Sci. USA 101:2747-2751.
  • Suzuki, Kazushi, Paul Babitzke, Sidney R. Kushner, and Tony Romeo. (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, Alexander V., Helen Yakhnin, and Paul Babitzke (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, Alexander V., Helen Yakhnin and Paul Babitzke (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, Paul, Carol S. Baker, and Tony Romeo. (2009) Regulation of translation initiation by RNA binding proteins. Ann. Rev. Microbiol. 63:27-44.
  • Yakhnin, Alexander V., and Paul Babitzke (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, Adrianne N., Laura M. Patterson-Fortin, Christopher A. Vakulskas, Jeffrey W. Mercante, Katarzyna Potrykus, Daniel Vinella, Martha I. Camacho, Joshua A. Fields, Stuart A. Thompson, Dimitris Georgellis, Michael Cashel, Paul Babitzke, and Tony Romeo. (2011) Circuitry linking the Csr and stringent response global regulatory systems. Mol. Microbiol. 80:1561-1580.
  • Martínez, Luary C., Helen Yakhnin, Martha I. Camancho, Dimitris Georgellis, Paul Babitzke, José L. Puente, and Victor H. Bustamante. (2011) Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD. Mol. Microbiol. 80:1637-1656.
  • Yakhnin, Helen, Alexander V. Yakhnin, Carol S. Baker, Elena Sineva, Igor Berezin, Tony Romeo and Paul Babitzke. (2011) Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by Es70 and EsS, and indirect transcriptional activation by CsrA. Mol. Microbiol. 81:689-704.
  • Mukherjee, Sampriti, Helen Yakhnin, Dave Kysela, Josh Sokoloski, Paul Babitzke and Daniel B. Kearns. (2011) CsrA-FliW interaction governs flagellin homeostasis and a checkpoint on flagellar morphogenesis in Bacillus subtilis. Mol. Microbiol. 82:447-461.
  • Yakhnin, Helen, Carol S. Baker, Igor Berezin, Michael A. Evangelista, Alisa Rassin, Tony Romeo, and Paul Babitzke. (2011) CsrA represses translation of sdiA, which encodes the N-acylhomoserine-L-lactone receptor of Escherichia coli, by binding exclusively within the coding region of sdiA mRNA. J. Bacteriol. 193:6162-6170.
  • Pannuri, Archana, Helen Yakhnin, Christopher A. Vakulskas, Adrianne N. Edwards, Paul Babitzke and Tony Romeo. (2012) Translational repression of NhaR, a novel pathway for multi-tier regulation of biofilm circuitry by CsrA. J. Bacteriol. 194:79-89.
  • Yakhnin, Alexander V., Helen Yakhnin and Paul Babitzke. (2012) Gel mobility shift assays to detect protein-RNA interactions. Methods Mol. Biol. 905:201-211.
  • Mukherjee, Sampriti, Paul Babitzke and Daniel B. Kearns. (2013) FliW and FliS function independently to control cytoplasmic flagellin levels in Bacillus subtilis. J. Bacteriol. 195:297-306.
  • Romeo, Tony, Christopher A. Vakulskas, and Paul Babitzke. (2013) Posttranscriptional regulation on a global scale: Form and function of Csr/Rsm systems. Env. Microbiol. 15:313-324.
  • Yakhnin, Alexander V., Carol S. Baker, Christopher A. Vakulskas, Helen Yakhnin, Igor Berezin, Tony Romeo and Paul Babitzke. (2013) CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage. Mol. Microbiol. 87:851-866.
  • Patterson-Fortin, Laura M., Christopher A. Vakulskas, Helen Yakhnin, Paul Babitzke, and Tony Romeo. (2013) Dual posttranscriptional regulation via a cofactor-responsive mRNA leader. J. Mol. Biol. 425:3662-3677.
  • Yakhnin, Alexander V., and Paul Babitzke. (2014) NusG/Spt5: are there universal functions of this ubiquitously conserved transcription elongation factor? Curr. Opin. Microbiol. 16:68-71.
  • Vakulskas, Christopher A., Archana Pannuri, Diana Cortés-Selva, Paul Babitzke, and Tony Romeo. (2014) Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures. Mol. Microbiol. 92:945-958.
  • Vakulskas, Christopher A., Anastasia H. Potts, Paul Babitzke, Brian M. Ahmer, and Tony Romeo (2015) Regulation of bacterial virulence by Csr (Rsm) systems. Microbiol. Mol. Biol. Rev. 79:193-224.
  • Abbott, Zachary D., Helen Yakhnin, Paul Babitzke, and Michele S. Swanson. (2015) csrR, a paralog and direct target of CsrA, promotes Legionella pneumophila resilience in water. MBio 6:e00595.
  • Yakhnin, Helen, and Paul Babitzke. (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, Hongmarn, Helen Yakhnin, Michael Connolly, Tony Romeo and Paul Babitzke. (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, Smarajit, Alexander V. Yakhnin, Aswathy Sebastian, Istvan Albert, and Paul Babitzke. (2016) NusA-dependent transcription termination prevents misregulation of global gene expression. Nature Microbiol. 1:15007. (doi:10.1038/nmicrobiol.2015.7)
  • Leng , Yuanyuan, Christopher A. Vakulskas, Tesfalem R. Zere, Bradley S. Pickering, Paula I. Watnick, Paul Babitzke, and Tony Romeo. (2016) Regulation of CsrB/C sRNA decay by EIIAGlc of the phosphoenolpyruvate: carbohydrate phosphotransferase system. Mol. Microbiol. 99:627-639.
  • Yakhnin, Alexander V., Katsuhiko S. Murakami, and Paul Babitzke. (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.
  • Chowdhury, Nityananda, Brian W. Kwan, Louise C. McGibbon, Paul Babitzke, and Thomas K. Wood. (2016) Toxin MqsR cleaves single-stranded mRNA with various 5' ends. MicrobiologyOpen 5:370-377.
  • Vakulskas, Christopher A., Yuanyuan Leng, Hazuki Abe, Takumi Amaki, Akihiro Okayama, Paul Babitzke, Kazushi Suzuki, and Tony Romeo (2016) Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins. (Nucleic Acids Res., in press)
  • Mukerjee, Sampriti, Reid Oshiro, Helen Yakhnin, Paul Babitzke, and Daniel B. Kearns. (2016) FliW antagonizes CsrA by a novel non-competitive allosteric mechanism. (Proc. Natl. Acad. Sci. USA, in press).