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.

Human immunodeficiency virus type 1 reverse transcriptase protects sugar moieties of a model template-primer DNA in a region from positions +3 to -15 from hydroxyl radical attack. A protected region of equivalent size migrates in concert with the translocating enzyme, as shown by hydroxyl radical footprints of replication complexes after primer extension by 4, 10, and 19 nt. The pattern of these footprints suggests that the DNA template-primer is in the A conformation when complexed with reverse transcriptase. Enhanced accessibility of the DNA template strand around position -15 to hydroxyl radicals indicates a conformational change in the template induced by the C-terminal RNase H-containing domain of p66 reverse transcriptase.Inhibition of human immunodeficiency virus reverse transcriptase (HIV RT) remains one of the most significant approaches in the fight against acquired immunodeficiency syndrome (AIDS). Combination therapy, using various RT inhibitors, has been discussed as a possible means of enhancing drug tolerance and reducing problems of drug resistance (1-4). Information regarding the structure of the retroviral polymerase would be an obvious advantage when attempting to design inhibitors with improved efficacy. A recent series of publications dealing with the structure of HIV-1 RT with enhanced resolution serves to emphasize this view. A neutron solution scattering study (5) has provided information about the spatial arrangement of the RT domains, and a crystal structure analysis of an RT-antibody complex at 0.35-nm resolution (6) has revealed the arrangement of the active sites within RT. Although recent publication of a structure model of Kohlstaedt et al. (7) at 0.3-nm resolution is sufficiently detailed to be considered useful for designing anti-RT drugs, there is little information about the structure of RT in complex with its RNA and DNA templates. The x-ray structure analysis of Arnold et al. (6) included template DNA, which was allowed to diffuse into the RT crystal, giving an approximate position ofthe template. However, the accuracy of the data was insufficient to determine whether the template DNA was in the A or B conformation. Kohlstaedt et al. (7) placed a template in the A conformation into the structure of isolated RT and presented a rather detailed picture of the complex. However, no study to date has revealed information on HIV RT during polymerization events, to our knowledge.As a complementary approach to crystallographic analyses, we have used hydroxyl radical footprinting (8) We have used hydroxyl radicals to determine contact points between a duplex DNA template-primer on which RT-catalyzed DNA synthesis was "arrested" at several positions along the template by incorporation of a chainterminating dideoxynucleoside triphosphate (ddNTP). Such complexes would be a reasonable approximation of DNAdependent DNA polymerase activity catalyzed by the retroviral polymerase during (+)-or second-strand synthesis (13). This approach has allowed us to study interactions with both ...

The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated. The occupancies of DNA fragments carrying the Al promoter of bacteriophage 17 were analyzed as a function of the length of flanking sequences adjacent to the promoter. Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy. This was studied quantitatively by using a set of DNA fragments with four identical Al promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV. The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy. Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion.Location of a DNA signal sequence by facilitated diffusion (1-4) has been proposed for a number of sequence-specific binding proteins. For example, there is good evidence in the cases of lac repressor (5, 6), EcoRI restriction endonuclease (7,8), and DNA polymerase (9) for mechanisms that compress the three-dimensional diffusion volume. Such mechanisms involve formation of a nonspecific protein-DNA complex and subsequent diffusion of the protein within or along the DNA domain until either the signal sequence is located or dissociation occurs. Translocation within the DNA domain results in a positionally uncorrelated search pattern, where the protein either dissociates from the DNA microscopically and probes adjacent sites on the DNA ("hopping") (4), or dissociates macroscopically and rebinds to a new site. This paper addresses two questions: (i) to what extent do one-dimensional diffusion processes participate in the location of a promoter and (ii) is this process directional? Sliding as a means of accelerating the promoter-search process by DNA-dependent RNA polymerase (EC 2.7.7.6) was proposed in 1982 by von Hippel et al. (10). We have designed a system that allowed us to test this idea by means of competition experiments in which the occupancies of promoters with flanking DNA sequences of different lengths were compared. Our hypothesis was that nonspecific DNA sequences serve as "antennae" (10) along which the Escherichia coli RNA polymerase moves to the promoter. If this antenna effect exists, the occupancy of a promoter should be influenced by the length of the flanking sequences. tests (17). The concentration of the DNA was determined by UV absorption (assuming an extinction coefficient at 260 nm of 20 cm2/mg). EXPERIMENTAL PROCEDURESMethods. Binding of RNA polymerase (3 ,AM) to promotercarrying DNA fragments (3 ,...

A series of RNA synthesizing transcription complexes, initiated at the T7 A1 promoter and halted at specific base positions ranging from +12 to +40, were analyzed by footprinting techniques; exonuclease III was used to determine the position of the bound RNA polymerase on the DNA and hydroxyl radicals were used to visualize the protein‐‐DNA contact sites within the protected areas. In the binding (open) complex without RNA there are two DNA‐domains, differing in their protection pattern. The first, extending from position +18 to ‐13, termed ‘melting domain’, is fully protected, whereas the second, extending from ‐14 to ‐55, termed ‘recognition domain’, shows only partial protection. At this domain, RNA polymerase is attached to one side of the DNA only, as indicated by the 10‐bp periodicity of the protection pattern. Our data show that the formation of a mature RNA transcribing complex is characterized by dissociation of the RNA polymerase from the recognition domain, whereby the size of the melting domain remains constant. This process is accomplished if the nascent RNA has reached a length of 11 bases. As the RNA reaches a length of 20 bases, the size of the melting domain decreases from approximately 30 to 23 bp. Further RNA synthesis leaves the protection pattern essentially unchanged. These data demonstrate that the formation of a mature RNA transcribing complex can be described by at least two transitions.

No abstract