New insights into the mechanism of heat shock response activation.

Heat shock (HS) response is a universal mechanism of protection against adverse environmental conditions. It is manifested mainly by rapid and robust induction of molecular chaperones and other cytoprotective proteins. In higher eukaryotes the activation of the HS response is mediated by a master regulator, heat shock transcription factor 1 (HSF1). Here we outline recent progress in understanding the early steps in HSF1 activation by heat in the context of existing models of HSF1 regulation.

Control of intrinsic transcription termination by N and NusA: the basic mechanisms.

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.

Isolation and characterization of sigma(70)-retaining transcription elongation complexes from Escherichia coli.

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.

An autocatalytic mechanism of protein nitrosylation.

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.

The mechanism of intrinsic transcription 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.

Transcription elongation: structural basis and mechanisms.

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.

Spatial organization of transcription elongation complex in Escherichia coli.

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.

The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase.

An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage.

Transcription processivity: protein-DNA interactions holding together the elongation complex.

The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the beta' subunit and the R interaction, to the COOH-terminal catalytic domain of the beta subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

Coupling between transcription termination and RNA polymerase inchworming.

Advancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism.

Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.

Discontinuous mechanism of transcription elongation.

During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site. The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region. However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed. Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control.

Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA.

Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.

Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli.

Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp). Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation. If expressed together, the four hsp are effective at physiological concentrations. Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins.