Faculty Bibliography

Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well

Transcription termination in Escherichia coli is controlled by many factors. The sequence of the DNA template, the structure of the transcript, and the actions of auxiliary proteins all play a role in determining the efficiency of the process. Termination is regulated and can be enhanced or suppressed by host and phage proteins. This complex reaction is rapidly yielding to biochemical and structural analysis of the interacting factors. Below we review and attempt to unify into basic principles the remarkable recent progress in understanding transcription termination and anti-termination

Nitric oxide (NO(.)) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological NO(.) concentrations, plasma albumin becomes saturated with NO(.) and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of NO(.) oxidation in the albumin hydrophobic core and specific transfer of NO(+) to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of NO(.) and its reactive oxides and controls the dynamics of NO(.)-dependant processes in the vasculature

Intrinsic transcription termination plays a crucial role in regulating gene expression in prokaryotes. After a short pause, the termination signal appears in RNA as a hairpin that destabilizes the elongation complex (EC). We demonstrate that negative and positive termination factors control the efficiency of termination primarily through a direct modulation of hairpin folding and, to a much lesser extent, by changing pausing at the point of termination. The mechanism controlling hairpin formation at the termination point relies on weak protein interactions with single-stranded RNA, which corresponds to the upstream portion of the hairpin. Escherichia coli NusA protein destabilizes these interactions and thus promotes hairpin folding and termination. Stabilization of these contacts by phage lambda N protein leads to antitermination

sigma(70) subunit is thought to be released from the core RNA polymerase (RNAP) upon the transition from initiation to elongation or shortly afterward. Here, we identify a population of RNAP from E. coli that retains sigma(70) throughout elongation. The relative amount of this population appears to depend on cellular growth and reaches its maximum during the stationary phase. The proportion of sigma(70)-retaining elongation complexes (EC-sigma(70)) is invariant with various promoters or distances from the transcription start site. EC-sigma(70) responds to pauses, intrinsic terminators, and the elongation factor NusA similarly to EC without sigma(70). However, EC-sigma(70) has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its profound role in gene expression and regulation in bacteria

Nitros(yl)ation is a widespread protein modification that occurs during many physiological and pathological processes. It can alter both the activity and function of a protein. Nitric oxide (( small middle dot)NO) has been implicated in this process, but its mechanism remained uncertain. ( small middle dot)NO is unable to react with nucleophiles under oxygen-free conditions, suggesting that its higher oxides, such as N(2)O(3), were actually nitrosylating agents. However, low concentrations and short lifespans of these species in vivo raise the question of how they could efficiently locate target proteins. Here we demonstrate that at physiological concentrations of ( small middle dot)NO, N(2)O(3) forms inside protein-hydrophobic cores and causes nitrosylation within the protein interior. This mechanism of protein modification has not been characterized, because all previously described mechanisms (e.g., phosphorylation, acetylation, ADP-ribosylation, etc.) occur via attack on a protein by an external modification agent. Oxidation of ( small middle dot)NO to N(2)O(3) is facilitated by micellar catalysis, which is mediated by the hydrophobic phase of proteins. Thus, a target protein seems to be a catalyst of its own nitrosylation. One of the applications of this finding, as we report here, is the design of specific hydrophobic compounds whose cooperation with ( small middle dot)NO and O(2) allows the rapid inactivation of target enzymes to occur

A ternary complex composed of RNA polymerase (RNAP), DNA template, and RNA transcript is the central intermediate in the transcription cycle responsible for the elongation of the RNA chain. Although the basic biochemistry of RNAP functioning is well understood, little is known about the underlying structural determinants. The absence of high- resolution structural data has hampered our understanding of RNAP mechanism. However, recent work suggests a structure-function model of the ternary elongation complex, if not at a defined structural level, then at least as a conceptual view, such that key components of RNAP are defined operationally on the basis of compelling biochemical, protein chemical, and genetic data. The model has important implications for mechanisms of transcription elongation and also for initiation and termination.

In bacteria, an intrinsic transcription termination signal appears in RNA as a hairpin followed by approximately eight uridines (U stretch) at the 3' terminus. This signal leads to rapid dissociation of the ternary elongation complex (TEC) into RNA, DNA, and an RNA polymerase. We demonstrate that the hairpin inactivates and then destabilizes TEC by weakening interactions in the RNA-DNA hybrid-binding site and the RNA-binding site that hold TEC together. Formation of the hairpin is restricted to the moment when TEC reaches the point of termination and depends upon melting of four to five hybrid base pairs that follow the hairpin's stem. The U stretch-induced pausing at the point of termination is crucial, providing additional time for hairpin formation. These results explain the mechanism of termination and aid in understanding of how cellular factors modulate this process

During RNA synthesis in the ternary elongation complex, RNA polymerase enzyme holds nucleic acids in three contiguous sites: the double-stranded DNA-binding site (DBS) ahead of the transcription bubble, the RNA-DNA heteroduplex-binding site (HBS), and the RNA-binding site (RBS) upstream of HBS. Photochemical cross-linking allowed mapping of the DNA and RNA contacts to specific positions on the amino acid sequence. Unexpectedly, the same protein regions were found to participate in both DBS and RBS. Thus, DNA entry and RNA exit occur close together in the RNA polymerase molecule, suggesting that the three sites constitute a single unit. The results explain how RNA in the integrated unit RBS-HBS-DBS may stabilize the ternary complex, whereas a hairpin in RNA result in its dissociation