The DNA polymer d(GC)n . d(GC)n can undergo a transition from the usual right-handed 10.4 base pairs (bp) per turn B form to a novel left-handed 12 bp per turn Z form in response to altered environmental conditions. Several other alternating purine-pyrimidine DNA polymers with modified bases have been shown to undergo transitions from B to Z conformations, with varying degrees of difficulty. We report here that the unmodified DNA polymer d(TG)n . d(CA)n readily undergoes a transition to a Z conformation when subjected to unwinding torsional stress in ionic conditions that are close to physiological. By using a two-dimensional gel electrophoresis system, we have determined both the critical free energy of supercoiling that is required to initiate the transition and the free energy of supercoiling that is required to maintain this polymer in the Z form.

We present evidence that excision of the nonreplicative transposon Tn10 involves three distinct chemical steps, first-strand nicking, hairpin formation, and hairpin resolution. This three-step mechanism makes it possible for a single protein-active site to cleave two DNA strands of opposite polarity, as appears to be the case in this reaction. We infer the existence of alternating bifunctionality within the active site with suitable modulation of substrate components between steps. DNA double-strand breaks are also made by a "hairpin mechanism" in V(D)J recombination, possibly reflecting the same basic constraints faced in the Tn10 system. Similarities in the basic chemical steps in Tn10 transposition and V(D)J recombination suggest that the V(D)J mechanism may have evolved from a bacterial transposition system.

No abstract

It is increasingly apparent that the genetic code is not static and that organisms use variations in the genetic code for selective advantage. Berg...

No abstract

Summary Hfq is a critical component of post‐transcriptional regulatory networks in most bacteria. It usually functions as a chaperone for base‐pairing small RNAs, although non‐canonical regulatory roles are continually emerging. We have previously shown that Hfq represses IS 10/Tn10 transposase expression through both antisense RNA‐dependent and independent mechanisms. In the current work, we set out to define the regulatory role of Hfq in the absence of the IS 10 antisense RNA. We show here that an interaction between the distal surface of Hfq and the ribosome‐binding site of transposase mRNA (RNA‐IN) is required for repressing translation initiation. Additionally, this interaction was critical for the in vivo association of Hfq and RNA‐IN. Finally, we present evidence that the small RNA ChiX activates transposase expression by titrating Hfq away from RNA‐IN. The current results are considered in the broader context of Hfq biology and implications for Hfq titration by ChiX are discussed.

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P 3408 A, S 3463 A, and SRR 3413-3415 AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of $7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR 3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein.Tra1-SRR 3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR 3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

A 34 base pair tract of the simple repeating dinucleotide d(AT)n-d(AT)n cloned into a 2.4 kb polylinker plasmid vector undergoes a structural transition in response to negative superhelical coiling. The transition has been characterized by 2 dimensional gel electrophoresis, mapping of S1, P1 and T7 endonuclease 1 sensitive sites, and mapping of sites that are sensitive to modification by bromoacetaldehyde. After S1 nuclease treatment it is possible to trap supercoiled species that are nicked on one or both strands near the center of the palindrome. These data show that the alternate state adopted by the d(AT)n-dAT)n insert is a cruciform rather than a Z conformation. Unlike other B-cruciform transitions the transition in d(AT)n-d(AT)n has a low activation energy and the transition is facilitated by the presence of magnesium ions. Evidence from in-vivo topoisomer distributions is presented which shows that under conditions of blocked protein synthesis the d(AT)n-d(AT)n insert will spontaneously adopt the cruciform state in-vivo in E. coli.