Biomedical subjects

R S Fuller

Publications and source records attributed to R S Fuller.

41 records · Page 3Linked to original sources

PubMed1984

The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites.

Specific binding of dnaA protein to the E. coli origin of replication (oriC) is essential for initiation of chromosomal replication. Based on binding of plasmid DNA fragments, dnaA protein recognizes in addition to oriC a number of specific sites: within or near the replication origins of pSC101, pBR322, and ColE1; within the regulatory regions of the dnaA and "X-protein" genes; and in IRL-Tn5. All strong binding sites share a common 9 bp sequence, 5'-TTATCACACAA, repeated at four conserved positions within oriC. As shown by DNAase I footprinting, dnaA protein binding is highly cooperative, covering 250 bp at oriC and 100 bp at single 9 bp sequence sites. Consistent with filter-binding and nuclease-protection studies, complexes of 20 to 30 dnaA monomers are visualized at oriC and other sites by electron microscopy. The functions of dnaA complexes and 9 bp sequences at these sites are discussed.

Bacterial Proteins↗

PubMed1983

Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.

Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.

Bacterial Proteins↗

PubMed1982

Enzymatic replication of E. coli chromosomal origin is bidirectional.

A soluble enzyme system has been discovered which specifically recognizes and replicates plasmids containing the Escherichia coli chromosomal origin, oriC. Electron microscopy has shown that plasmid replication begins at or near oriC from which it progresses bidirectionally to completion. Control of initiation of a cycle of chromosomal replication and mechanisms of priming and fork movement can now be explored using this system.

Chromosomes, Bacterial↗

PubMed1981

Enzymatic replication of the origin of the Escherichia coli chromosome.

An enzyme system that replicates plasmids bearing the origin of the Escherichia coli chromosomes (oriC) has the following physiologically relevant features. The system (i) depends completely on low levels of exogenously furnished supercoiled oriC plasmids, (ii) uses only those plasmids that contain the intact oriC region of about 245 base pairs, (iii) initiates replication within or near the oriC sequence and proceeds bidirectionally, (iv) proceeds linearly, after a 5-min lag, for 30-40 min to produce as much as a 40% increase over the input DNA, (v) depends on RNA polymerase and gyrase as indicated by total inhibition by rifampicin and nalidixate, (vi) depends on replication proteins (e.g., dnaB protein and single-stranded DNA binding protein) as judged by specific antibody inhibitions, (vii) operates independently from protein synthesis, and (viii) depends on dnaA activity, as suggested by the inactivity of enzyme fraction from each of two dnaA temperature-sensitive mutant strains, and complementation (with a 15-fold overproduction of complementing activity) by a fraction from a strain containing the dnaA gene cloned in a multicopy plasmid. Resolution and analysis of factors that control the initiation of a chromosome cycle should become accessible through its enzyme system.

Bacterial Proteins↗

PubMed1978

The attenuator of the tryptophan operon in E.coli: rho-mediated release of RNA polymerase from a transcription termination complex in vitro.

In vivo, termination of transcription at the attenuator site of the tryptophan (trp) operon of E. coli is influenced by the protein termination factor rho. In vitro, termination does not depend on rho factor, and is very efficient in a purified system consisting only of RNA polymerase, the DNA template, nucleoside triphosphates, and buffer. The extent of termination in this system is unaffected over a wide range of salt and nucleoside triphosphate concentration. However, there is a 10-fold stimulation of trp leader mRNA synthesis if rho factor is present during the transcription reaction. This stimulation occurs only at low molar ratios of polymerase to template, and can be blocked by rifampicin. It is thus most likely due to the recycling of RNA polymerase molecules that have been released from the attenuator site by rho factor. In fact, transcription of the trp leader region in vitro results in the fomration of a stable termination complex which can be observed on sucrose gradients or by binding to nitrocellulose filters. These data indicate that a major function of rho at the trp attenuator is to release completed transcripts from a pre-formed termination complex, rather than to cause the cessation of elongation.

Base Sequence↗