Comparison of fluorescence optical respirometry and microbroth dilution methods for testing antimicrobial compounds.

An analysis of the usefulness of the fluorescence optical respirometry test method to study several antimicrobials was performed. An oxygen-sensitive sensor: ruthenium-tris(4,7-diphenyl-1,10-phenanthroline) dichloride (Ru(DPP)3Cl2), the phosphorescence of which is quenched by molecular oxygen, was synthesised according to a method modified by us and then applied. A prototype sensitive measurement system was designed and constructed. Analyses of the impact of various antimicrobial chemical factors were performed: ampicillin, co-trimoxazole, nystatin, and newly synthesised compounds. It was shown that optical respirometry allows for analysis of the culture growth kinetics of bacteria and fungi and determination of cell growth parameters. It was shown also that MIC values obtained by fluorescence optical respirometry are consistent with the results of the MIC determinations made by serial dilution method (traditional MIC testing using CLSI). The method allows the time to obtain results to be significantly reduced (from 24-48 h to 5-7 h for bacteria and 24 yeasts) and allows the effect of concentrations below the MIC for the metabolic activity of microorganisms to be monitored. The sensitivity of the method allowed the volume of the tested samples to be lessened from 160 µl to 50 µl. Fluorescence optical respirometry allows for the rapid detection and evaluation of the action of various chemical compounds on the metabolic activity of microorganisms in real-time measurement of fluorescence intensity.

A novel collection of accessory factors associated with yeast RNA polymerase II.

A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II. However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J. Kim et al., 1994, Cell 77, 599-608; A. Koleske and R. Young, 1994, Nature 368, 466-469). By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme. The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p). Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression. Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X. Shi et al., Mol. Cell. Biol., 1996 16, 669-676). The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.

Nucleation of RNA chain formation by Escherichia coli DNA-dependent RNA polymerase.

We have studied the early steps in RNA synthesis. The kinetic behaviour of the nascent RNA, having chain lengths between 3 and 11 bases, and the transcription fidelity were analysed using the bacteriophage T7 A1 promoter. By moving the stop-inducing base at position +12 in the wild-type template in single base steps upstream, a set of closely related templates was constructed which allowed stalling of the complexes in the registers 11, 10, 9 and 8. Using this set of templates sigma-factor release was determined. It occurs when RNA synthesis has proceeded to base position +9. Analysis of the RNA synthesis both with and without heparin yielded the following results: there are three kinds of complexes, (a) the well-known abortively transcribing complex, which is present until the RNA has reached a length of 5 bases, (b) an intermediate complex having RNA chain lengths between 6 and 8 bases, which is stably bound but has high forward as well as back reaction rates, (c) complexes with RNA chains consisting of more than 8 bases, which are stably bound and do not contain the sigma-factor. In general, the likelihood of chain elongation and the stability of the complexes increases with increasing RNA chain length in the early stages of RNA synthesis. Also the transcription fidelity increases correspondingly. Lack of fidelity leads to additional RNA products during the abortive state of transcription. "Read through" of RNA polymerase at stall positions of +8 to +11 also result from misincorporation.

Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase.

Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.

Topography of intermediates in transcription initiation of E.coli.

Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.

DNA-dependent RNA polymerase of Escherichia coli induces bending or an increased flexibility of DNA by specific complex formation.

Escherichia coli RNA polymerase is shown to induce bending or an increased flexibility of the promoter DNA. This is a specific effect of holoenzyme (core enzyme and sigma-factor). The centre of the flexibility is 3 bp upstream of the initiation point of RNA synthesis. This flexibility or bending is maintained during RNA synthesis by core enzyme.