Transcription mapping of the Escherichia coli chromosome by electron microscopy

French, Sarah L.; Miller, O. L.

doi:10.1128/jb.171.8.4207-4216.1989

Cited by 80 publications

(87 citation statements)

References 73 publications

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“…Direct observation by transmission electron microscopy showed that at least 65 RNAPs are present on a single operon and that multiple operons are expressed coordinately (French and Miller 1989). Thus, our observation that the promoter region is necessary and sufficient for colocalization of rRNA operons invited the hypothesis that active transcription by RNAPs was responsible.…”

Section: Dna Movement Could Account For Part Of the Apparent Distancesupporting

confidence: 53%

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

et al. 2016

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The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180°on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus.

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Section: Dna Movement Could Account For Part Of the Apparent Distancesupporting

confidence: 53%

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

et al. 2016

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“…The rrn operons are identified on the chromosome as sites of high transcriptional activity with a characteristic spatial arrangement of RNA polymerase molecules and nascent rRNA transcripts, suggestive of a double Christmas tree, that is easy to recognize (9). Figure 2 shows electron micrographs of an rRNA operon from each of the two nus mutant strains as well as a wild-type control for comparison.…”

Section: Resultsmentioning

confidence: 99%

Transcriptional Polarity in rRNA Operons of Escherichia coli nusA and nusB Mutant Strains

et al. 2005

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Synthesis of ribosomes in Escherichia coli requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of rrn operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on rrn operons were determined for each mutant. Compared to the wild type, the 16S gene in the nusB mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the nusA mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their rrn operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5-to 3-fold for the nus mutant strains over that for wild-type cells. Thus, strains carrying either a nusA mutation or a nusB mutation have defects in transcription of 23S rRNA.In the synthesis of rRNA in Escherichia coli, RNA polymerase undergoes several rrn operon-specific modifications that double the transcription elongation rate and allow readthrough of Rho-dependent terminators (1,7,29,30,32). The modification events require specific nucleotide sequences in the leader and spacer regions of the rrn operons as well as interacting proteins that alter the properties of the transcribing polymerase. Thus, profound changes in the basic capabilities of RNA polymerase occur when rrn operons are transcribed, but these changes and their consequences have yet to be clearly described. The nucleotide sequence signals leading to the changes in RNA polymerase activity in rrn operon transcription are called BoxB, BoxA, and BoxC in the leader sequence and BoxB and BoxA in the spacer region between the 16S and 23S genes. BoxA is a conserved 12-nucleotide sequence (Fig. 1), BoxB is a conserved stem-loop structure, and BoxC is a GT-rich region (3, 19). These sequences are related to those used in the bacteriophage lambda N protein antitermination system (8, 12). Protein factors involved in rrn antitermination (rRNA-AT) are less well defined, but they likely include the N utilization substances (Nus) NusA, NusB, NusE (ribosomal protein S10), NusG, and ribosomal protein S4 (15,23,24,27,29,30,32). In vivo studies have documented the importance of NusB and NusG for rRNA-AT and, in conjunction with BoxA, for increasing the rate of elongation by RNA polymerase (28, 32). NusG, NusE (S10), and S4 are all essential proteins. It was assumed that NusB would also be an essential cellular protein, but in studies with transport protein mutants, two...

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“…Northern (RNA) blots, performed as previously described (39), revealed an RNA band accumulating just below the position of 23S RNA upon induction with IPTG (data not shown). The size of this band corresponds to the expected size of a pUV17-specific rm transcript from which the 16S RNA part has been removed by cotranscriptional processing, as for the native rm transcripts (10,13).…”

Section: Resultsmentioning

confidence: 99%

The RNA chain elongation rate in Escherichia coli depends on the growth rate

1994

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We determined the rates of mRNA and protein chain elongation on the lacZ gene during exponential growth on different carbon sources. The RNA chain elongation rate was calculated from measurements of the time elapsing between induction of lacZ expression and detection of specific hybridization with a probe near the 3' end of the mRNA. The elongation rate for the transcripts decreased 40%o when the growth rate decreased by a factor of 4, and it always correlated with the rate of translation elongation. A similar growth rate dependency was seen for transcription on the infB gene and on a part of the rrnB gene fused to a synthetic, inducible promoter. However, the untranslated RNA chain specified by the rrnB gene was elongated nearly twice as fast as the two mRNA species encoded by infB and lacZ.The rate of RNA chain elongation in Escherichia coli is generally regarded as being constant and independent of the growth rate, while it is well accepted that the polypeptide chain growth rate depends on the medium (5). It is known that decoupling of translation from transcription causes premature mRNA chain termination in several genes and operons (1) and that a tight coupling between RNA polymerase and the leading ribosome is able to suppress transcriptional pausing and termination at attenuators of amino acid and nucleotide biosynthetic genes (16,19,20,29). Since premature transcription termination does not seem to prevail during steady-state growth, one might expect that rates of transcription and translation were very tightly adjusted to each other in steadystate cultures. Therefore, we wanted to measure the transcription elongation rate during exponential growth in different media. MATERIALS AND METHODSBacterial strains and plasmids. Strain MAS90 is E. coli K-12 thi Apro-lac recAl/F' lacIqP lacZ::Tn5 proAB+ and was previously described (39). As used here, MAS90 was transformed with different plasmids, pMAS2 (34), pUV12, pUV14, and pUV17 (37), which all encode resistance to ampicillin.Growth conditions. The experimental cultures were started by dilution of exponentially growing cultures in the same medium. Cultures were grown in shaking Erlenmeyer flasks at 37°C. The medium was either Luria-Bertani (LB) (24) containing 100 ,ug of ampicillin per ml (doubling time, 24 min) or the A+B salt medium of Clark and Maal0e (7). This salt medium was supplemented with ampicillin (100 ,ug/ml) and thiamine (1 pug/ml) and one Preparation of RNA and dot blot analysis. For determination of the RNA chain growth kinetics, 4-ml samples were withdrawn for RNA purification at 10-s intervals after induction of transcription as described by Vogel et al. (39). The RNA samples were analyzed by dot blot hybridization on GeneScreen membranes with different radioactive RNA probes (37). In short, 5 ,ug of RNA was dissolved in 50% deionized formamide containing 6% formaldehyde, denatured, and applied on a dot blot manifold. The RNA was fixed to the membrane by UV irradiation for 2 min and prehybridized without drying. Prehybridization, hybridizati...

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Transcription mapping of the Escherichia coli chromosome by electron microscopy

Cited by 80 publications

References 73 publications

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

Transcriptional Polarity in rRNA Operons of Escherichia coli nusA and nusB Mutant Strains

The RNA chain elongation rate in Escherichia coli depends on the growth rate

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