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The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage depends on the function of the viral transcriptional regulatory protein, Tat, which increases the efficiency of RNA elongation (transcriptional processivity) in response to cellular activation. To quantify levels of Tat functionin vivo,a quantitative competitive RT-PCR assay was developed that reflects levels of TAR leader fragments (nonprocessive transcripts) and viral mRNA (processive transcripts), indicating low or high levels of Tat function, respectively. The abundance of these RNA species was measured in peripheral blood mononuclear cells (PBMC) of 22 HIV-1-positive individuals (CD4+T cell counts 63–934/mm3) and in established cell line models of HIV constitutive replication (H9IIIB) and reversible latency (U1 and ACH-2). In PBMC, the level of total viral transcripts ranged over four orders of magnitude; however, nonprocessive transcription predominated: 70% of PBMC samples had a ratio of processive to total transcripts of

Transcription of bacteriophage SP01 middle promoters is specifically initiated by a complex of the Bacillus subtilis host's RNA polymerase core (E) with the SP01 gene 28 transcription-regulating protein, gp28. Normal SP01 DNA contains hydroxymethyluracil (hmUra) in place of thymine and E.gp28 preferentially transcribes hmUra-containing DNA. Hybrid DNA molecules containing an SP01 middle promoter, PM25.1, have been constructed in which one DNA strand contains T and the other hmUra. The major feature of these reciprocal hybrid promoters is that one has, predominantly, T substituted for hmUra in the central −35 recognition sequence in the transcribed strand, while the other has, predominantly, T substituted for hmUra in the −10 recognition sequence in the non-transcribed strand. Binding by the E.gp28 RNA polymerase and transcription of these hybrid promoters and of the normal, all-hmUra, promoter have been compared. Both hybrid promoters are weaker than the normal PM25.1 promoter, but the hybrid promoter with T substituted in the −10 sequence is the weakest of the set. The DNase I footprint of the normal PM25.1 promoter shows temperature-dependent protection of a relatively long stretch of DNA downstream from the transcriptional start site, correlating with a thermal transition of transcriptional activity of promoter complexes. The stronger of the hybrid promoters also undergoes this transition, but the weaker does not. We discuss these findings in terms of protein-DNA interactions determining specificity for a modified nucleotide at this promoter.

The bacteriophage SPO1 gene 28-encoded protein, gp28, directs specific recognition of viral middle promoters in hydroxymethyluracil-containing DNA by the Bacillus subtilis host's RNA polymerase core. Using appropriately sensitive methods of detection, we have shown that discrimination against thymine-containing DNA is not absolute and that the gp28-containing RNA polymerase precisely initiates transcription at two thymine-containing SPO1 middle promoters.

SPO1 gene 28, which codes for the positive regulator of middle transcription, gp28, has been mapped by in vitro DNA-directed protein synthesis to a segment located between 89.5 and 91.5 kbp from the left end of the genome. Gene 27 is also located in this interval. One early promoter has been mapped to this region of the genome (P E 13 at 90.1 kbp) and early transcription of gene 28 could initiate there. Two other early promoters, P E 14 and P E 15, are located at 99.2 and 112.5 kbp from the left end. These three early promoters are significantly weaker in vitro than those early promoters which are located in the terminal repeat. The functions associated with P E 14 and P E 15 are unknown, but evidence is presented that P E 15 is utilized in vivo . Thirteen middle promoters have also been mapped within the region of the genome extending from 85 to 113.5 kbp. The middle promoters are interspersed with the three early promoters and in some cases, yield overlapping early and middle transcription. Such overlapping early and middle transcription units apparently generate at least some em class SPO1 RNA in vivo .

The genome of the Bacillus subtilis phage SP01 contains a 12.6-kilobase-pair terminal redundancy. The transcription units in this region, which are utilized in vitro by B. subtilis RNA polymerase holoenzme, have been mapped. In vitro transcripts were separated and characterized by gel electrophoresis. Since there is such a large number of promoters in this region, it was necessary to employ conditions of transcription under which only subsets of all the transcripts would be made. Selective synthesis was achieved by transcription of restriction fragments containing a small number of promoters and by dinucleotide priming. Transcription units were mapped by observing the shortening of transcripts that resulted from restriction enzyme cleavage of the template. The mapping indicates the presence of two blocks of overlapping transcription units, oriented so as to yield convergent transcription toward a bidirectional termination region. The frequency of readthrough at the bidirectional terminator is low. Several partial termination sites of differing efficiencies have also been identified, including two that are active only at low ribonucleoside triphosphate concentrations. We also describe an effect of NaCl concentration on the migration of RNA in gels, which is specific for only two of the SP01 early transcripts. This effect may indicate unusual secondary structures for these two RNA molecules.

Summary The bulk transfer-RNA of Bacillus subtilis contains approximately one thymine residue per molecule. The methyl group of the thymine does not become labeled when the organism is grown in the presence of [methyl-14C]methionine, although other methylated bases of the B. subtilis tRNA do become labeled.