Domain architecture of a high mobility group A-type bacterial transcriptional factor.

Myxococcus xanthus transcriptional factor CarD participates in carotenogenesis and fruiting body formation. It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins. The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities. We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure. CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts. As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD. The acidic region, in addition to modulating DNA binding, confers structural stability to CarD. We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M. xanthus.

Folding kinetics of phage 434 Cro protein.

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.

Thermodynamic analysis of the structural stability of phage 434 Cro protein.

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi29 early-late transcriptional switch.

Protein p6 is a nonspecific DNA-binding protein occurring in high abundance in phage phi29-infected cells. Here, we demonstrate a novel role for this versatile histone-like protein: its involvement in regulating the viral switch between early and late transcription. p6 performs this role by exhibiting a reciprocal functional interaction with the regulatory protein p4, also phage encoded, which is required for repression of the early A2b and A2c promoters and activation of the late A3 promoter. On the one hand, p6 promotes p4-mediated repression of the A2b promoter and activation of the A3 promoter by enhancing binding of p4 to its recognition site at PA3; on the other, p4 promotes p6-mediated repression of the A2c promoter by favoring the formation of a stable p6-nucleoprotein complex that interferes with RNA polymerase binding to PA2c. We propose that the observed interplay between proteins p6 and p4 is based on their DNA architectural properties.

Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.

The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head-on. However, head-on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery. Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase.

Bacteriophage phi29 DNA replication arrest caused by codirectional collisions with the transcription machinery.

The consequences on replication of collisions between phi29 DNA polymerase, a monomeric replicase endowed with strand displacement capacity, and the transcription machinery have been studied in vitro. Codirectional collisions with stalled transcription ternary complexes at four different promoters in the phi29 genome were found to block replication fork progression. Upon collision, the DNA polymerase remained on the template and was able to resume elongation once the RNA polymerase was allowed to move. Collisions with RNA polymerase molecules moving in the same direction also interfered with replication, causing a decrease in the replication rate. These results lead to the proposal that in bacteriophage phi29 a transcription complex physically blocks the progression of a replication fork. We suggest that temporal regulation of transcription and the low probability that the replication and transcription processes colocalize in vivo contribute to achieving minimal interference between the two events.

Saccharomyces cerevisiae mutants defective in plasmid-chromosome recombination.

We have studied the recombinational repair of a double-strand break (DSB) in a plasmid-borne ade2::HO-site by an intact ade2 allele following the induction of a galactose-inducible GAL-HO gene. If GAL-HO expression is not attenuated by the presence of a low level of glucose in the galactose medium, deleterious effects are observed. Our comparison of the effects of several rad mutations on the relative efficiencies of DSB repair at both the ade2::HO-site and at the chromosomal MAT locus indicate that the two processes share common functions. Not surprisingly, most of the recombination-defective mutants found using our assay are alleles of genes in the RAD52 epistasis group. The recombination and repair deficiencies vary among the different mutant groups and also among mutants within a group. In general, there is a correlation between the extents of the recombination and repair defects. Our screen also turned up a novel rfa1 allele with a pronounced deficiency in DSB repair and recombination and a srs2 mutation which causes only a mild defect.

A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes.