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We show that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large (150-500 bp) loops. These G-loops are formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other. G-loop formation requires a G-rich nontemplate strand and reflects the unusual stability of the rG/dC base pair. G-loops and G4 DNA form efficiently within plasmid genomes transcribed in vitro or in Escherichia coli. These results establish that G4 DNA can form in vivo, a finding with implications for stability and maintenance of all G-rich genomic regions.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] catalyzes the addition and regeneration of the 3'-terminal CCA sequence of tRNAs. We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the "top half" of tRNA-the acceptor stem and TpsiC stem-loop of tRNA. CCA addition to the top half tDNA minihelices requires a 2' as well as a 3' OH at the 3' terminus of the tDNA. Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TpsiC loop. These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.

We describe the purification, cloning, and characterization of the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] from the thermophilic archaebacterium, Sulfolobus shibatae. Characterization of an archaeal CCA-adding enzyme provides formal proof that the CCA-adding activity is present in all three contemporary kingdoms. Antibodies raised against recombinant, expressed Sulfolobus CCA-adding enzyme reacted specifically with the 48-kDa protein and fully depleted all CCA-adding activity from S. shibatae crude extract. Thus, the cloned cca gene encodes the only CCA-adding activity in S. shibatae. Remarkably, the archaeal CCA-adding enzyme exhibits no strong homology to either the eubacterial or eukaryotic CCA-adding enzymes. Nonetheless, it does possess the active site signature G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][CLIVMFY]3-5 of the nucleotidyltransferase superfamily identified by Holm and Sander (1995, Trends Biochem Sci 20:345-347) and sequence comparisons show that all known CCA-adding enzymes and poly(A) polymerases are contained within this superfamily. Moreover, we propose that the superfamily can now be divided into two (and possibly three) subfamilies: class I, which contains the archaeal CCA-adding enzyme, eukaryotic poly(A) polymerases, and DNA polymerase beta; class II, which contains eubacterial and eukaryotic CCA-adding enzymes, and eubacterial poly(A) polymerases; and possibly a third class containing eubacterial polynucleotide phosphorylases. One implication of these data is that there may have been intraconversion of CCA-adding and poly(A) polymerase activities early in evolution.

In nuclear extracts of primary murine B lymphocytes cultured with LPS we have identified an inducible DNA-binding activity that is a candidate regulator of isotype-switch recombination. This LPS-responsive factor, which we refer to as LR1, is induced in LPS-cultured primary cells with kinetics that parallel isotype-switch recombination. LR1 binds sequences from the S gamma 1, S gamma 3, and S alpha switch regions, as well as the heavy-chain enhancer, and these binding sites define a consensus that occurs in each of the murine switch regions. LR1 activity is present in pre-B and B-cell lines but absent from primary B cells that have not been cultured with mitogen and from highly differentiated B-cell lines. LR1-binding activity depends on phosphorylation and is lost following incubation of nuclear extracts with acid phosphatase. The LPS inducibility and phosphorylation dependence of LR1 activity suggest that this factor monitors kinase-dependent events in cell development and communicates them to the chromosome. The locations of its binding sites and the kinetics of its induction are consistent with a role for LR1 in regulation of isotype switching.