An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon

Hartmann, Roland K.; Ulbrich, Norbert; Erdmann, Volker A.

doi:10.1016/0300-9084(87)90009-5

Cited by 50 publications

(51 citation statements)

References 22 publications

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“…To analyze whether our preparation yielded the functional holoenzyme, we carried out an in vitro transcription using two T. thermophilus templates with defined single promoters, for which the in vivo starts of transcription have been determined. One DNA fragment, 0.3 kb in size, carried the promoter of a 23S/5S rRNA operon from T. thermophilus [8], and the second fragment, 2.2 kb in size, carried the promoter of a protein gene of unknown function whose in vivo start of transcription was identified as well by nuclease S1 mapping (data not shown). As can be seen in Fig.…”

Section: Resultsmentioning

confidence: 98%

“…3 B, lanes 4-8) more as a gel-overloading effect than due to transcript heterogeneity. In addition, we used the identical T. thermophilus DNA fragments as templates for a commercially available E. coli RNA polymerase preparation, since both T. thermophilus promoters were shown to be functional in E. coli by electronmicroscopic binding studies (data not shown) and induction of a promoter-deficient chloramphenicol acetyltransferase gene [8] (and data not shown). However, with the E. coli RNA polymerase preparation, a less discrete transcript pattern was observed (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Calf thymus DNA, spermidine [N-(3-aminopropyl)-l,4-butanediamine], polymin P (polyethylenimine), ampicillin and chloramphenicol were purchased from Sigma. Thermine [N,N'-bis ( 3 -aminopropyl [8] or a 2.2-kb fragment with a T. thermophilus protein gene promoter (unpublished data), were grown at 37°C in Luria broth in the presence of ampicillin and chloramphenicol (each 50 mg/l). Gel electrophoreses of nucleic acids and proteins were performed as described .…”

Section: Methodsmentioning

confidence: 99%

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Isolation and physical properties of the DNA‐directed RNA polymerase from Thermus thermophilus HB8

Wnendt¹,

Hartmann²,

Ulbrich³

et al. 1990

European Journal of Biochemistry

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The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for p and /l', 40 kDa for CI and 92 kDa for u. The RNA polymerase is active at elevated temperatures (65°C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in viva starts of transcription were characterized by nuclease S1 mapping.The properties of RNA polymerases derived from eubacterial and archaebacterial sources have been reviewed [l]. A large-scale preparative method employing four chromatography steps has been described for the isolation of the RNA polymerase from Thermus thermophilus [2]. The method yielded two kinds of RNA polymerase with different properties.Eubacterial RNA polymerases display a strongly conserved subunit composition and can be isolated as a holoenzyme (~~' c I~) o and as a core enzyme which lacks the u subunit. The different subunits have molecular masses of the following ranges: p and p' 140-190 kDa; CI about 40 kDa; cr in Gram-positive bacteria of 40-60 kDa or of about 70-90 kDa in Gram-negative bacteria. Recent results provided direct evidence that the u subunit is responsible for promoter site selection and that the factor dissociates after initiation of RNA synthesis [3]. The fact that different 0 subunits switch on specific sets of genes during the sporulation process of Bacillus subtilis further emphasizes the keyrole of the u subunit in transcriptional initiation [4].As part of this project, we reported before on the organization of rDNA in T. thermophilus and its regulatory elements governing transcription [5 -91. Here we present a new method for the isolation of the RNA polymerase and describe some properties of the enzyme. MATERIALS AND METHODS MaterialsEnzymes and molecular mass marker proteins were purchased from Boehringer Mannheim, Pharmacia and Bethesda Research Laboratories. Radioactive nucleotides were from Amersham. Calf thymus DNA, spermidine [N-(3-aminopropyl)-l,4-butanediamine], polymin P (polyethylenimine), ampicillin and chloramphenicol were purchased from Sigma. Thermine [N,N'-bis( 3 -aminopropyl)-1,3-diaminopropane] wasCorrespondence to V. A. Erdmann, Freie Universitat Berlin, lnstitut fur Biochemie, Thielallee 63, D-1000 Berlin 33Enzymes. Restriction endonucleases BamHI and SmaI (EC 3.1.21.4); DNA-directed RNA polymerase (EC 2.7.7.6).from Aldrich. Microdialysis of samples (representing Superose 12 or Mono Q fractions) for SDS gel electrophoresis was performed employing Millipore VFPF filters. Trichloroacetic-acid-precipitated nucleic acids were collected on Whatman GF/A filters. MethodsThermus thermophilus HB8 (ATCC 27634) cells were grown at 70 -75 "C in medium D, as described previously [lo], supplemented pe...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and physical properties of the DNA‐directed RNA polymerase from Thermus thermophilus HB8

Wnendt¹,

Hartmann²,

Ulbrich³

et al. 1990

European Journal of Biochemistry

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“…Mycoplasma hyopneumoniae contains a single copy of 16S-23S and a separate 5S gene (45). Pirellula marina, a planctomycete, contains two separate copies of both 23S-5S and 16S (23), and Thennus thermophilus has a similar number and arrangement of rRNA genes (11,12). There also appears to be no close linkage among the two copies of the 23S and 16S rDNAs and the one copy of the 5S rDNA in Leptospira interrogans (9).…”

Section: Resultsmentioning

confidence: 99%

Genome maps of Campylobacter jejuni and Campylobacter coli

et al. 1992

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Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.

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“…Because the mutations in the 3Ј end of the O-16S sequence differentiate the translational initiation sites of natural and O-ribosomes, production of the O-ribosome requires the synthesis, processing, and incorporation of O-16S rRNA into 70S ribosomes (25,26). With a single characterized exception (27)(28)(29), ribosomal RNA is produced in a single transcript (25). This transcript generally contains a 5Ј leader sequence, the 16S rDNA, a spacer that may contain a tDNA, the 23S rDNA, and the 5S rDNA with or without additional tDNAs.…”

Section: Synthesis Of a Transcription-translation Fflmentioning

confidence: 99%

Synthesis of orthogonal transcription-translation networks

Chin

2009

Proc. Natl. Acad. Sci. U.S.A.

119

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Orthogonal, parallel and independent, systems are one key foundation for synthetic biology. The synthesis of orthogonal systems that are uncoupled from evolutionary constraints, and selectively abstracted from cellular regulation, is an emerging approach to making biology more amenable to engineering. Here, we combine orthogonal transcription by T7 RNA polymerase and translation by orthogonal ribosomes (O-ribosomes), creating an orthogonal gene expression pathway in Escherichia coli. We design and implement compact, orthogonal gene expression networks. In particular we focus on creating transcription-translation feed-forward loops (FFLs). The transcription-translation FFLs reported cannot be created by using the cells' gene expression machinery and introduce information-processing delays on the order of hours into gene expression. We refactor the rRNA operon, uncoupling the synthesis of the orthogonal 16S rRNA for the O-ribosome from the synthesis and processing of the rest of the rRNA operon, thereby defining a minimal module that can be added to the cell for O-ribosome production. The minimal O-ribosome permits the rational alteration of the delay in an orthogonal gene expression FFL. Overall this work demonstrates that system-level dynamic properties are amenable to rational manipulation and design in orthogonal systems. In the future this system may be further evolved and tuned to provide a spectrum of tailored dynamics in gene expression and investigate the effects of delays in cellular decisionmaking processes. directed evolution ͉ orthogonal ribosome ͉ protein engineering ͉ synthetic biology

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An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon

Cited by 50 publications

References 22 publications

Isolation and physical properties of the DNA‐directed RNA polymerase from Thermus thermophilus HB8

Isolation and physical properties of the DNA‐directed RNA polymerase from Thermus thermophilus HB8

Genome maps of Campylobacter jejuni and Campylobacter coli

Synthesis of orthogonal transcription-translation networks

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