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.
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.
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.