2The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing growth regimens. Before they can transcribe from specific DNA promoter sequences, bacterial core RNA polymerases (with subunit composition ␣ 2 ␤␤Ј) must combine with a dissociable sigma subunit () to form RNA polymerase holoenzyme (␣ 2 ␤␤Ј). Since the discovery of factors (6), it has become clear that these proteins are central to the function of the RNA polymerase holoenzyme. The reversible binding of alternative factors allows formation of different holoenzymes able to distinguish groups of promoters required for different cellular functions. In addition to double-strand DNA promoter recognition and binding, proteins are closely involved in promoter melting (e.g., references 31,36,49,51,74,76,128), inhibit nonspecific initiation, are targets for activators, and control early transcription through promoter clearance and release from RNA polymerase (48,49,53). Here we describe the functioning of the bacterial 54 -RNA polymerase that is the target for sophisticated signal transduction pathways (103) involving activation via remote enhancer elements (5, 95).Based on structural and functional criteria, the different factors identified in bacteria can be grouped in two classes, one of which has a single member, 54 . Many factors belong to the 70 class, the major factor which is involved in expression of most genes during exponential growth (72). 54 (also called N ) differs both in amino acid sequence and in transcription mechanism from the 70 class (80). Despite the lack of any significant sequence similarity, both types of bind the same core RNA polymerase. Nonetheless, they produce holoenzymes with different properties.With the recognition that the 54 protein represented an entirely new class of factor, what had once been regarded as an aspect of transcription restricted to higher organisms became a well-established feature of certain bacterial regulatory systems, particularly those associated with nitrogen metabolism. Activation of 54 -RNA polymerase employs specialized bacterial enhancer-binding proteins whose activating function requires nucleotide hydrolysis (94,96,122) (Fig. 1). In this system, initiation rates are controlled via regulation of the DNA melting step that is necessary for establishing the open promoter complex (85,94,97). Bacterial enhancer-dependent transcription can be studied with just two purified proteins (an activator and the 54 -RNA polymerase holoenzyme) and the appropriate DNA template, facilitating progress in understanding mechanistic aspects of 54 functioning. Below we review the biology and biochemistry of the 54 -RNA polymerase.

High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter. The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence. Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide. Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase. Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively. In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added. Nevertheless, oligonucleotide synthesis persists under all conditions tested. Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total. This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond. As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release. These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro. Production of a long RNA transcript is then essentially an escape from this cycling reaction. The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reaction.

Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation. Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro. Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16. Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex. Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.

The supercoiling levels of plasmid DNA were determined from Escherichia coli which was grown in ways that are known to alter global patterns of gene expression and metabolism. Changes in DNA supercoiling were shown to occur during several types of these nutrient upshifts and downshifts. The most dramatic change in supercoiling was seen in starved cells, in which two populations of differentially relaxed plasmids were shown to coexist. Thus, some changes in the external nutritional environment that cause the cells to reorganize their global metabolism also cause accompanying changes in DNA supercoiling. Results of experiments with dinitrophenol suggested that the observed relaxations were probably not due to reduced pools of ATP. When rifampin was used to release supercoils restrained by RNA polymerase, the cellular topoisomerases responded by removing these new, unrestrained supercoils. We interpret these results as implying that the cellular topological machinery maintains a constant superhelical energy in the DNA except during certain growth transitions, when changes in metabolism and gene expression are accompanied by changes in DNA supercoiling.The negative supercoiling of DNA is essential to the maintenance of normal cell function, especially in such cellular processes as transcription, replication, and recombination (for reviews, see reference 7 and 38). Several Escherichia coli proteins exist that have the ability to alter the level of supercoiling by breaking and rejoining DNA (for a review, see reference 41). Presently, the known enzymes are DNA gyrase (11), which can use ATP to increase negative supercoiling, and topoisomerases I (40) and III (4), which can relax negatively supercoiled DNA. Since supercoiling in vivo is maintained by the opposing actions of these enzymes (5,29,30), their relative activities could be regulated to produce various levels of supercoiling. If such a change in supercoiling were to occur in response to physiological stimuli, it could contribute to the global regulation of cellular processes. In this study we investigated the extent to which the level of DNA supercoiling varies during growth of E. coli, with emphasis on how these changes could be involved in the global regulation of transcription.While the importance of DNA supercoiling in the maintenance of cellular processes has been established, changes in DNA supercoiling which could account for global regulatory responses remain to be demonstrated. There are several examples in which plasmids isolated from bacteria grown under different conditions have been reported to vary in the level of supercoiling, including cells grown at different temperatures (12) and cells grown to the stationary phase (34). It has been shown in additional studies (27,28) that the level of supercoiling of plasmids has some dependence on the local DNA sequence. One special case of control has been reported in which the level of chromosomal supercoiling differed in Salmonella typhimurium growing aerobically and anaerobically because of changes in ...

In vivo "footprints" of the ginA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. Ear54

No abstract

No abstract

No abstract