<P><B>Regulation of Transcription</B><P><I>Intracellular signal levels and promoter activities.</I>Transcription of many genes is regulated by combinations of multiple signals. In <I>Escherichia coli</I>, combinatorial control is typical in the case of operons related to utilization of different sugars in the absence of glucose. To understand regulation of the transport and metabolic pathways in the galactose system, we measured activities of the six gal regulon promoters simultaneously, using an <I>in vitro</I>transcription system containing purified components. Input functions were computed on the basis of the experimental measurements. We observed four different shapes of input functions. From the results, we can conclude that the structure of the regulatory network is insufficient for the determination of signal integration. It is the actual structure of the promoter and regulatory region, the mechanism of transcription regulation, and the interplay between transcription factors that shape the input function to be suitable for adaptation.</P><P><I>Optical configuration of the inducer D-Galactose.</I>The two optical forms of aldohexose galactose differing at the C-1 position, alpha-D-galactose and beta-D-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of D-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other D-galactose-metabolizing enzymes are coded by genes that constitute an operon in <I>Escherichia coli</I>. The operon is repressed by the repressor GalR and induced by D-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of D-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of D-galactose. Here we report <I>in vivo</I>and <I>in vitro</I> experiments showing that both alpha-D-galactose and beta-D-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the alpha configuration inactivate the induction capacity of the sugar, the effect of substitutions in the beta configuration varies depending upon the nature of the substitution;methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.</P><P><I>DNA sequences that override transcription elongation block.</I>The DNA loop that represses transcription from galactose (gal) promoters is infrequently formed in stationary-phase cells because the concentration of the loop architectural protein HU is significantly low at that state, resulting in expression of the operon in the absence of the gal inducer D-galactose. Unexpectedly, transcription from the gal promoters under these conditions overrides physical block because of the presence of the Gal repressor bound to an internal operator (O(I)) located downstream of the promoters. We have shown here that although a stretch of pyrimidine residues (UUCU) in the RNA:DNA hybrid located immediately upstream of O(I) weakens the RNA:DNA hybrid and favors RNA polymerase (RNAP) pausing and backtracking, a stretch of purines (GAGAG) in the RNA present immediately upstream of the pause sequence in the hybrid acts as an antipause element by stabilizing the RNA:DNA duplex and preventing backtracking. This facilitates forward translocation of RNAP, including overriding of the DNA-bound Gal repressor barrier at O(I). When the GAGAG sequence is separated from the pyrimidine sequence by a 5-bp DNA insertion, RNAP backtracking is favored from a weak hybrid to a more stable hybrid. RNAP backtracking is sensitive to Gre factors, D-galactose, and antisense oligonucleotides. The ability of a native DNA sequence to override transcription elongation blocks in the gal operon uncovers a previously unknown way of regulating gal metabolism in <I>Escherichia coli</I>. It also explains the synthesis of gal enzymes in the absence of inducer for biosynthetic reactions.</P><P><I>Dominant negative autoregulation.</I>Many transcription factors repress transcription of their own genes. Negative autoregulation has been shown to reduce cell-cell variation in regulatory protein levels and speed up the response time in gene networks. In this work we examined transcription regulation of the galS gene and the function of its product, the GalS protein. We observed a unique operator preference of the GalS protein characterized by dominant negative autoregulation. We show that this pattern of regulation limits the repression level of the target genes in steady states. We suggest that transcription factors with dominant negative autoregulation are designed for regulating gene expression during environmental transitions.</P><P><I>Function of nucleoid protein HU.</I>We have previously proposed that HU confers specific structures to the nucleoid and the structure determines its gene expression profile. To test that we have done DNA tiling arrays of <I>E. coli</I>strains with various HU mutants, and we are in the process of analyzing the data. We also note that most actions described for HU protein are by binding to nucleoid DNA for chromosome compaction. We designed an experiment to test the hypothesis that HU has additional functions, yet to be determined, by binding to RNA. In order to test directly the <I>in vivo</I>binding of HU to <I>E. coli</I>RNA, we preformed RNA immuno-precipitation (RIP) Chip. A strain containing eight myc tags fused to the chromosomal HUalpha was constructed. The protein was functional. We also constructed an additional strain, deleted for HUbeta. The new <I>E. coli</I>strain [8myc-HUalpha delHUbeta] was able to support phage Mu growth indicating that the fusion protein is active. Next, the 8myc tag was used to pool (IP) HUalpha from logarithmic growing cells and RNA purification was performed on the IP sample. The RNA isolated was analyzed by hybridizing directly to <I>E. coli</I>tilling arrays. These results showed that HU binds to all tRNA (except two) and rRNA, 4 ncRNA, 29 mRNA fragments. These results suggested a role of HU in RNAfunctions, presumably in translation. To test this hypothesis, we identified the mRNA targets of HU binding using two approaches:</P><P>1. Comparison of protein expression profiles of the wild-type and HU deficient- strain (delhupA delhupB) by 2D-gel electrophoresis, followed by mass-spectrometry identification of selected protein spots.</P><P>2. Analysis of structural similarity between HU and other RNA binding proteins using DALI database.</P><P>Using the first approach, we identified proteins whose expression was significantly altered in HU-deficient mutant relative to the wild-type. We selected spots that were either absent or significantly increased in delhupA delhupB relative to the wild-type strain. Mass spectrometry analysis revealed two tar [summary truncated at 7800 characters]
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