Faculty Bibliography

Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that uses a riboswitch to control a metabolic feedback loop through differential processing of the precursor RNA 3' terminus. When cellular thiamin pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product, and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa, suggesting that this mechanism is active since vascular plants emerged 400 million years ago

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures

Recent discoveries of RNA-based regulatory mechanisms have prompted substantial interest in how they formed and the extent to which varying environmental conditions have influenced their evolution. One class of RNA-based regulatory mechanism that has been found in bacteria is the riboswitch, regulating the biosynthesis of certain vitamins by an RNA genetic control clement that senses small molecules and responds with a structural change that affects transcription termination or translation initiation without the participation of proteins. By taking the thiamin pyrophosphate (TPP)-riboswitch in Bacillus subtilis as a model system, we wish to examine whether beneficial mutations may exist at the level of RNA that will cause an improvement in organism fitness. By computationally analyzing the difference in primary and secondary structure of the B. subtilis TPP-riboswitch collected from the xeric 'African' south-facing slope (SFS) vs. the mesic, 'European', north-facing slope (NFS) in 'Evolution Canyon' III at Nahal Shaharut, southern Israel, we wish to experimentally study the environmental effect on transcription termination in these RNA-based regulatory mechanisms that are believed to be of ancient origin in the evolutionary time scale. Computational results, so far, indicate that specific mutations affect the riboswitch conformation by causing a global rearrangement. We would like to check whether such mutations could be adaptive mutations that may have a beneficial fitness effect, taking the TPP-riboswitch as a model system at the micro-scale. Empirical results so far indicate that in the promoter region of the TPP-riboswitch, all mutations increase nucleotide GC content in the xeric SFS, whereas in the mesic NFS they increase AT content. Preliminary examination of termination efficiency of strains found exclusively on one slope or the other, reveal increased termination efficiency in the presence of TPP and at more moderate temperatures, but only a suggestion of greater termination efficiency from strains found on both slopes. We expect that further results will shed light on the mutational differences of TPP-riboswitch sequences found on opposite slopes of 'Evolution Canyon' III at Nahal Shaharut, potentially leading to interesting discoveries that relate to the topic of adaptive, nonrandom mutations

Large noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Recent reports identify a functional lncRNA, Evf-2, that operates during development to control the expression of specific homeodomain proteins, and they provide important insights into the mechanism of cooperation between a newly discovered nuclear receptor co-repressor protein (SLIRP) and steroid receptor activator RNA. Evf-2 is the first example of lncRNA directly involved in organogenesis in vertebrates

Bacterial core RNA polymerase (RNAP) must associate with a sigma factor to recognize promoter sequences. Escherichia coli encodes seven sigma factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of sigma32, the heat-shock sigma factor, and estimate that there are 120-150 sigma32 promoters in total. Unexpectedly, 25% of these sigma32 targets are located within coding regions, suggesting novel regulatory roles for sigma32. The majority of sigma32 promoter targets overlap with those of sigma70, the housekeeping sigma factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPsigma70 and RNAPsigma32 initiating transcription in vitro with similar efficiency and from identical positions. SigmaE-regulated promoters also overlap extensively with those for sigma70. These results suggest that extensive functional overlap between sigma factors is an important phenomenon

Riboswitches are common cis-acting regulatory elements in bacteria. They are made of nascent RNA that changes its conformation in response to direct binding of cognate metabolites. The publication of five high-resolution crystal structures provides a comprehensive view of how riboswitches sense their ligands and points to new challenges in this emerging field

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as 'punctuation marks' for DNA replication in vivo

The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation

We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA-DNA, DNA-RNA, and RNA-RNA base pairing associated with (i) the translocational and size fluctuations of the transcription bubble; (ii) changes in the associated DNA-RNA hybrid; and (iii) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process

Numerous sophisticated systems have been described that protect bacteria from increased levels of reactive oxygen species. Although indispensable during prolonged oxidative stress, these response systems depend on newly synthesized proteins, and are hence both time and energy consuming. Here, we describe an 'express' cytoprotective system in Bacillus subtilis which depends on nitric oxide (NO). We show that NO immediately protects bacterial cells from reactive oxygen species by two independent mechanisms. NO transiently suppresses the enzymatic reduction of free cysteine that fuels the damaging Fenton reaction. In addition, NO directly reactivates catalase, a major antioxidant enzyme that has been inhibited in vivo by endogenous cysteine. Our data also reveal a critical role for bacterial NO-synthase in adaptation to oxidative stress associated with fast metabolic changes, and suggest a possible role for NO in defending pathogens against immune oxidative attack