An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase

Ridley, S P; Oeschger, Max P.

doi:10.1128/jb.152.2.736-746.1982

J Bacteriol

1982

DOI: 10.1128/jb.152.2.736-746.1982

|View full text |Cite

An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase

S P Ridley¹,

Max P. Oeschger²

Abstract: An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-se… Show more

Help me understand this report

Search citation statements

Order By: Relevance

Paper Sections

Select...

Citation Types

Supporting

Mentioning

Contrasting

Year Published

1990

2005

Publication Types

Select...

Article5

Relationship

Self Cite0

Independent5

Authors

Journals

Cited by 5 publications

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Isolation of chromosomal mutations that affect carotenoid production inEscherichia coli: mutations alter copy number of ColE1-type plasmids

Luan

Jackson

Rouvière

et al. 2005

FEMS Microbiology Letters

View full text Add to dashboard Cite

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.

show abstract

Isolation of chromosomal mutations that affect carotenoid production inEscherichia coli: mutations alter copy number of ColE1-type plasmids

Luan

Jackson

Rouvière

et al. 2005

FEMS Microbiology Letters

View full text Add to dashboard Cite

show abstract

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Severinov¹,

Mooney²,

Darst³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the ␤ and ␤-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. Here, we show that in Escherichia coli the two genes can be fused and that the resulting 2751-amino acid ␤::␤ fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro. The results establish that the C terminus of the ␤ subunit and the N terminus of the ␤ subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivo may occur co-translationally. DNA-dependent RNA polymerase (RNAP)1 is the central enzyme of gene expression and a major target for regulation. Cellular RNAPs are large, multisubunit protein complexes. Core RNAP of Escherichia coli (ϳ380 kDa) contains four polypeptides: ␤Ј (155 kDa), ␤ (150 kDa), and a dimer of ␣ (37 kDa). Core RNAPs from eukaryotes contain 12 or more subunits with a total mass in excess of 600 kDa (1). The subunit composition of archaeal, chloroplast, and some eubacterial RNAPs is even more complex due to splits in the genes coding for the largest subunits (2, 3). Despite these differences, all cellular RNAPs exhibit striking and co-linear sequence similarities with the bacterial subunits. The two largest subunits comprise 60% of the RNAP mass and appear principally responsible for most of the enzyme's functions.The synthesis of RNAP subunits is coordinately regulated (4), but the exact mechanisms are unknown. In eukaryotes, many genes coding for RNAP subunits have similar regulatory elements, suggesting coordinated expression (5). In eubacteria and archaea, genes coding for the ␤-and ␤Ј-like subunits are organized in an operon; the gene coding for the ␤-like subunit always precedes that coding for the ␤Ј-like subunit (6). In eubacteria, the two genes are separated by a short, untranslated linker; in archaea they overlap by several codons (7).In this work we demonstrate that the E. coli rpoB and rpoC genes, coding, respectively, for the 1342 amino acid-long ␤ and the 1407-amino acid-long ␤Ј subunits, can be fused and that the resulting ␤::␤Ј fusion protein assembles into functional RNAP in vivo and in vitro. Furthermore, when a hexahistidine tag (His 6 tag) is inserted at the fusion site between ␤ and ␤Ј, the resulting RNAP can be immobilized on a sorbent containing Ni 2ϩ ions and is transcriptionally active in the immobilized state. On the basis of these data we conclude that the C terminus of ␤ and the N terminus of ␤Ј are within a very short distance of each other on the surface of the RNAP molecule during all phases of transcription.

show abstract

Mutational Analysis of β′260–309, a ς70 Binding Site Located on Escherichia coliCore RNA Polymerase

Arthur¹,

Anthony²,

Burgess³

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

In eubacteria, the subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the ␤ subunit that interacts with 70 in vitro. This region of ␤ contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260 -309 of ␤, to look at disruption of the 70 -core interaction. Several of the mutants were defective for binding 70 in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant ␤ is the sole source of ␤ subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for E 70 holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999) Cell 98, 811-824), the region homologous to ␤ 260 -309 of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil.

show abstract

scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.

Contact Info

customersupport@researchsolutions.com

10624 S. Eastern Ave., Ste. A-614

Henderson, NV 89052, USA

This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.

Blog Terms and Conditions API Terms Privacy Policy Contact Cookie Preferences Do Not Sell or Share My Personal Information

Made with 💙 for researchers

Part of the Research Solutions Family.

An amber mutation in the gene encoding the beta' subunit of Escherichia coli RNA polymerase

Cited by 5 publications

References 43 publications

Isolation of chromosomal mutations that affect carotenoid production inEscherichia coli: mutations alter copy number of ColE1-type plasmids

Isolation of chromosomal mutations that affect carotenoid production inEscherichia coli: mutations alter copy number of ColE1-type plasmids

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Mutational Analysis of β′260–309, a ς70 Binding Site Located on Escherichia coliCore RNA Polymerase

Contact Info

Product

Resources

About