Fig. 1. RNAP location/distribution and nucleoid structure in cells grown in different media. Images of the rpoC-gfp cells grown in rich medium LB (top row), in glucose-minimal medium (middle row) or in LB medium plus the antibiotic rifampicin (bottom row). The first column is an overlay of images of cells and their nucleoids. The compactness of the nucleoid is reflected by the relative amount of the cytoplasmic space in the cell. Note that the cytoplasmic space is apparent in the cell grown in rich media as the nucleoids are compact, whereas the reverse is true for the cell treated with rifampicin inhibiting transcription. The middle column is composed of RNAP-GFP images. Note that the bright spots or transcription foci are evident only in a fast-growing cell cultured in rich media. The third column is an overlay of RNAP-GFP (pseudo colored green) and nucleoid images (pseudo colored red). Transcription foci become either orange or green spots in these overlay images.

Fig. 2. The effects of the stringent response and two E. coli mutations conferring different stringent response phenotypes on RNAP distribution and nucleoid structure. The panels in the figure are arranged the same as in Fig. 1. Cells of wild type, relA (relaxed mutant), and rpoB3449 (stringent RNAP mutant) were all grown in rich media; however, amino acid starvation was induced by the addition of serine hydroxamate (final concentration, 100 μg/ml for 30 min) in the center two rows.

Fig. 3. Normalized contrast of the RNAP-GFP fluorescence signal in nucleoids of wild type and mutant rpoC–gfp cells under different physiological conditions. The normalized contrast parameter was measured from 100 nucleoids. In this analysis, larger values of normalized contrast indicate a more heterogeneous distribution of RNAP within the nucleoids, which in turn is indicative of the presence of transcription foci. The labels across the top specify the relevant genetic markers of the strains used in this analysis. The labels at the bottom specify the different physiological conditions: LB, cells grown in LB medium; Glc-minimal, cells grown in glucose-minimal medium; A.A. starvation, cells grown in LB medium and after 20 min of treatment with serine hydroxamate [adapted from the study by (Cabrera and Jin, 2003)].

Fig. 4. Model linking stable RNA synthesis, RNAP distribution and the dynamic structure of the nucleoid. The E. coli chromosome is represented as blue lines folded in loops, the ori of replication as a black square, the seven rRNA operons as large red circles with letters, and two representative tRNA operons as small red circles. The RNAP molecules are represented as small green circles. For simplicity, only two putative transcription factories/foci, which make the nucleoid more compact by pulling different stable RNA operons into proximity, are indicated here (bottom part of the diagram, large green circles labeled 1 and 2) [adapted from the study by (Cabrera and Jin, 2003)].

Fig. 5. The transcription factories/foci appear to be located in the periphery of the nucleoid to facilitate the coupling between transcription and ribosomal assembly and/or translation. In (A), a pair of nucleoids (pseudo colored red) and RNAP-GFP with four transcription foci (pseudo colored green) in a fast-growing cell cultured in rich medium LB are shown. Superimposing the two images indicates that while the transcription foci 2 and 4 are clearly located in the periphery of the bulk nucleoid mass, the transcription foci 1 and 3 seem to be located within the nucleoids; however, those RNAPs apparently located within the nucleoid could in fact have been surrounding the nucleoid (B). If this is the case, the RNAP location would facilitate the coupling between transcription and translation and/or ribosome assembly (C). For simplicity, only one ribosome (blue color) translating an mRNA (red color) in one of the transcription foci [concentrated RNAP (green color)] is shown. In this case, three DNA loops (black color) located far way from each other in the genome are pulled into proximity by active transcription.

Relative synthesis of stable RNA and mRNA for amino acid biosynthetic operons in wild type and mutant strains under different growth conditions

The entries in the table are intended qualitatively only to show the trend of relative expression of stable RNA compared to that of mRNA for amino acid biosynthetic operons under different growth conditions. For wild type, values in other growth conditions are compared to that in rich media. For the two E. coli mutants which have altered the stringent response phenotypes, , ↑, and ↓ mean similar, increased or decreased RNA synthesis rate compared to that of the wild type strain in the same growth condition.