Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. III. High resolution thermal denaturation and circular dichroism studies.

T C Goodman,R D Klein,R D Wells

doi:10.1016/s0021-9258(18)33610-x

Abstract

High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments. The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC). A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs. This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments. The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R. D., and Wells, R. D. (1982) J. Biol. Chem. 257, 12962-12969). Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C. CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter. The presence of homopolymers had a marked effect on the CD spectra of the fragments.

Highlights

High resolution thermal denaturation and circular suggested that CAP binding could facilitate transcription by dichroism studies were performed on a series of six disrupting the DNA helix (1, 6, 7, and reviewed in Ref. 8)
The frag- Whereas this boundary is deleted in the molecules studied ments varied in size from 132 to 193 bp and contained Escherichia coli wild type and U V 5 lactose promoters both with and without homopolymer insertions of poly(&) poly(dT) and poly(dG) poly(dC)
The relatively larger effect of the at one-half maximum height (AT) sequence on the wild type promoter region may be correlated with the increased levels of in uitro transcription activity described in the preceding paper

Summary

Length of fraq ment

Fragment ends pRW30 pHW31 pRW47 pRW33 pRW40 pRW39 wt UV5 wt-AT UV5-AT wt-GC UV5-CC bP. After each series of CD spectrawere obtained, the absorption of the samples was measured using a Cary 219 specabcdef trophotometer. The further mathematical treatment of the dataincluding calculation of the average spectra and determination of the standard deviation between spectra was done as described previously (IO). Theoretical Calculations-The theoretical melting curves were calculated using the loop entropy model of the DNA helix-coil transition as described previously (16-18).The melting temperatures were. In producing the calculated CI) spectra, T-matrices 1 and 14 were generated as described previously (10, 19,201 fromthe CD spectra of repeating DNA polymers (9, 21). The calculated CD spectra of each of the fragments were derived from these T-matrices andthe known. Matrix 1 was employed because it included the CD spectra of the homopolymers, poly(dA).poly(dT) and poly(dG).polydC)

RESULTS

Experimental and theoretical values for the melting transitions

Fragment desienation

DISCUSSION