The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.

Among several subspecies of 30S subunits of Escheriehia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein Sicontaining subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein SI from Caulobacter crescentus substitutes for E. coli SI despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the SI-containing subspecies. Protein SI is lost by salt treatment of ribosomes.Initiation of protein synthesis in bacterial systems involves the binding of the 30S ribosomal subunit to specific regions of mRNA accompanied by binding of fMet-tRNA (for review, see ref. 1). Electrophoresis of ribosomes on agarose-polyacrylamide gels (2) has demonstrated that, depending on the method of isolation and dissociation of the ribosomes, 30S subunit preparations contain at least two major subspecies. The main difference between the two subspecies is the presence of ribosomal protein Si in the more slowly migrating form (3,4). It was previously shown (4) that 30S subunits containing protein SI form a weak, species-specific complex with MS2 RNA in the absence of initiation factors. Moreover, interference factor ia (5-7), a protein indistinguishable from protein Si (8, 9), restores the capacity of Si-deficient E. coli ribosomes to bind MS2 RNA, simultaneously converting the faster to the slower 30S species (10).In this communication we report on the further functional analysis of subspecies of 30S subunits. We find that only the SI-containing species participates in the formation of the 30S initiation complex with MS2 RNA as messenger. Both subspecies are active in the AUG-dependent ribosomal binding of fMet-tRNA and in the poly(U)-dependent binding of Phe-tRNA. We further show that protein Si from Caulobacter crescentus substitutes for E. coli Si despite the fact that C. crescentus 30S ribosomes do not bind coliphage RNA (1,Y 12). The subspecies composition of 30S subunit preparations obtained under various conditions is also described. MATERIALS AND METHODSRibosomes of E. coli Q13 and MRE 600 were prepared as described (4, 12). Conditions for the isolation of 30S subunits and for assays of their activity are given in the legends to figures and tables. Before use, the subunits were activated by warming for 5 min at 370 (13).Purification of E. coli protein S1 from the ribosomal wash was recently described (10). Nearly homogeneous S1 was also obtained from ribosomes by the procedure of Tal et al. (14), followed by chromatography on agarose. The two preparations were indistinguishable by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. C. crescentus S1 was isolated in the course of purification of C. crescentus 30S ribosomal proteins in Dr. H. G. Wittmann's laboratory (S. Leffler, unpublished). Briefly, the 1...

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The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.

(ATCC 19089) were laboratory strains; Bacillus brevis Vm4 was kindly provided by Z. Kurylo-Borowska, the Rockefeller University. Cells were grown as described (13,15,16) and harvested at mid-logarithmic growth phase.Ribosomes and Ribosomal Subunits. These were obtained by procedures established for E. coli, and are described in recent publications (12, 13). Unfractionated crude ribosomes were washed twice (15 hr and 4-5 hr) in a buffer [20 mM Trisacetate (pH 7.8), 10 mM magnesium acetate, 0.1 mM dithiothreitol] containing 1.0 M NH4C1. To ascertain whether the 30S ribosomal subunits were fully devoid of initiation 'factors, each batch of washed 70S ribosomes was assayed before fractionation into subunits (Table 1). It was particularly important to use IF-3-free ribosomes. As seen in Table 1,