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Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli . HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro . We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known.

Bacteriophage λ is a temperate bacteriophage, meaning that it can reproduce and develop either in a lytic or lysogenic state. When λ infects its bacterial host Escherichia coli , the phage may develop lytically, causing cell lysis with the release of hundreds of progeny virus, or it may abort

Two regulatory proteins, Gal repressor and isorepressor, control the expression of the gal and mgl operons in Escherichia coli. The transcription start sites for galR and galS, the genes for the repressor and isorepressor, were determined by primer extension of in vivo transcripts. Study of the promoter-lacZ gene fusions introduced into the chromosome indicated that galS expression was elevated in cells in which the normal galS gene was interrupted, but not in cells in which the galR gene was deleted. When both genes were disrupted, galS expression was further elevated. Expression from the galS promoter was stimulated by the addition of D-fucose, repressed by glucose, and dependent on cyclic AMP receptor protein (CRP). Expression of a similar gene fusion of the galR promoter to lacZ was unregulated. Both galR and galS genes contain two potential operator sites (OE and OI) and a CRP-binding site. The arrangement of OE, OI, and the CRP-binding site in the galS gene is analogous to the arrangement in the gal and mgl promoters, but the arrangement in galR is atypical. The increased concentration of the isorepressor when inducer is present may facilitate early shutoff of the isorepressor-regulated genes of the gal regulon when inducer (substrate) concentration falls.

In wild-type Escherichia coli, expression of the gal operon is negatively regulated by the Gal repressor and is induced 10- to 15-fold when the repressor is inactivated by an inducer. In strains completely deleted for galR, the gene which encodes the Gal repressor, the operon is derepressed by only 10-fold without an inducer. But this derepression is increased further by threefold during cell growth in the presence of an inducer, D-galactose or D-fucose. This phenomenon of extreme induction in the absence of Gal repressor is termed ultrainduction--a manifestation of further inducibility in a constitutive setup. Construction and characterization of gene and operon fusion strains between galE and lacZ, encoding beta-galactosidase as a reporter gene, show that ultrainduction occurs at the level of transcription and not translation. Transcription of the operon, from both the cyclic AMP-dependent P1 and the cyclic nucleotide-independent P2 promoters, is subject to ultrainduction. The wild-type galR+ gene has an epistatic effect on ultrainducibility: ultrainduction is observed only in cells devoid of Gal repressor protein. Titration experiments show the existence of an ultrainducibility factor that acts like a repressor and functions by binding to DNA segments (operators) to which Gal repressor also binds to repress the operon.

The two optical forms of aldohexose galactose differing at the C-1 position, α- d -galactose and β- 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 Escherichia coli . 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 in vivo and in vitro experiments showing that both α- d -galactose and β- 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 α configuration inactivate the induction capacity of the sugar, the effect of substitutions in the β configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.

We have studied the initiation of transcription of the gal operon in Escherichia coli (i) by analyzing the 5'-triphosphate ends and (ii) by measuring the level of promoter-proximal gal mRNA made in vivo. The 5' termini were identified and quantified by capping with GTP:mRNA guanyltransferase, and the mRNA levels were determined by hybridization of pulse-labeled [32P]RNA with a specific DNA probe. Our results conclusively demonstrate the in vivo activities of two promoters, P1 and P2, with separate initiation sites (S1 and S2) as suggested before from in vitro and in vivo experiments (S. Adhya and W. Miller, Nature [London] 279:492-494, 1979; R. E. Musso, R. DiLauro, S. Adhya, and B. de Crombrugghe, Cell 12:847-854, 1977). We have also studied the effect of cyclic AMP (cAMP) on in vivo gal transcription and found that whereas total gal transcription remains largely unchanged, the relative proportions of the S1 and S2 mRNAs are influenced by the level of cAMP in the cell. In strains devoid of cAMP (cya), transcription initiates equally at S1 and S2; in cAMP-proficient cells (cya+), the S1 initiation increases twofold with a concomitant decrease in S2 initiation. Addition of a saturating amount of exogenous cAMP to cya mutant cells results in a relatively larger switch from S2 to S1. Our results clearly show that while cAMP is an inhibitor of S2, it is not an absolute requirement for transcription initiation at S1, but only acts to increase low-level transcription from the P1 promoter. Using these approaches, we have also studied gal promoter mutants (P211, P18, and P35) which show altered behavior in transcription initiations and in response to cAMP. On the basis of these results, we have discussed models by which transcription initiates at the two overlapping gal promoters (P1 and P2) and discussed how cAMP level modulates the switch between them.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.

Expression of the EScherichia coli gal operon under the control of the prophage lambda promoter pL leads to gross discoordinacy of gal expression. Expression of the most promoter-distal cistron galK is much greater than expression of the promoter-proximal cistron galE. We had previously shown that transcription of the gal operon is coordinate after prophage induction. A survey of protein synthesized after prophage induction indicated that lack of expression of galE is due to a failure of translation of the galE sequence in the pL-gal transcript. This failure of translation of the galE sequence may be due to extensive dyad symmetry present in the vicinity of the gal promoter region of the pL-gal transcript. This symmetry could result in a ribonucleic acid stem-loop structure, blocking the attachment of ribosomes at the Shine-Dalgarno sequence of galE. To test this model, strains bearing the IS1 or IS2 insertion, deletion, or new promoter mutation within the symmetrical region were constructed. The restoration of some galE expression after such disruptions of the symmetrical region indicated that the ribonucleic acid stem-loop structure did play a role in the discoordinate expression of gal from pL. However, failure to obtain galE expression coordinated with high levels of galK expression suggested that other components were involved, perhaps other symmetries between galE and the pL transcript.