CDNA library construction for the Lambda ZAP-based vectors.

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 A resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.

Bacteriophage lambda↗

PubMed1997

Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates.

Several models have been proposed for the mechanism of transcript termination by Escherichia coli RNA polymerase at rho-independent terminators. Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991) Biochemistry 30, 1097-118) postulated that the transcription complex is stabilized by enzyme-nucleic acid interactions and the favorable free energy of a 12-bp RNA-DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex. To distinguish between these models, we have tested whether E. coli RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA. RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager-von Hippel model predicts that intrinsic termination will not occur. We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure. However, E. coli RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs. Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by E. coli RNA polymerase.

Bacteriophage lambda↗

PubMed1997

An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.

Bacteriophage lambda↗