Faculty Bibliography

The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase. The latest major advance was the high-resolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA. Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle. They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance

Transfection of retrovirus packaging cells with linear DNA from a retroviral vector missing the 3' long terminal repeat (3' LTR) results in production of infectious virus. Analysis of the newly formed proviruses indicates that restoration of the 3' LTR sequences necessary for reverse transcription and integration occurred due to end-to-end template switching by mammalian RNA polymerase II (RNAP II) in the packaging cells. These observations argue that RNAP II can utilize double-strand breaks and gaps in DNA to generate 'recombinant' transcripts in vivo and suggest a mechanism for mutation and recombination of retroviruses

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