A human cDNA encoding the small subunit of RNA polymerase II transcription factor SIII

A full-length cDNA encoding a human homolog of the 15-kDa subunit (p15) of RNA polymerase II elongation factor SIII was isolated and sequenced. Comparison of the open reading frames of the human p15 cDNA and the previously characterized rat p15 cDNA [Garrett et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5237-5241] indicates that they encode identical proteins and are 93% conserved in nucleotide sequence.     

A systemic small RNA signaling system in plants

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.     

The quorum-hindered transcription factor YenR of Yersinia enterocolitica inhibits pheromone production and promotes motility via a small non-coding RNA

The YenR and YenI proteins of Yersinia enterocolitica resemble the quorum sensing proteins LuxR and LuxI of Vibrio fischeri. Apo-YenR activated a gene, designated yenS, that lies adjacent to and divergent from yenR. YenR-dependent expression of yenS was inhibited by endogenous or exogenous 3-oxohexanoylhomoserine lactone (OHHL) a pheromone made by YenI. Purified apo-YenR bound non-cooperatively to two 20-nucleotide sites that lie upstream of yenS. Binding occurred in the absence of (OHHL), and YenR was largely released from the DNA by this pheromone. yenS encoded two non-translated RNAs 169 and 105 nucleotides long that share the same 5' end but have different 3' ends. One or both RNAs inhibited the translation and accumulation of the yenI mRNA by binding to a region that overlaps the YenI start codon. A mutation in yenI strongly stimulated swarming motility on the surface of semi-solid agar, while exogenous OHHL completely suppressed this phenotype. Hypermotility in yenI mutants was also suppressed by mutations in yenR or yenS, suggesting that YenS plays a direct, stimulatory role in swarming motility.     

Binding stoichiometry of an RNA aptamer and its transcription factor target

Ion-pair reverse-phase high-performance liquid chromatography is presented as a versatile platform for the rapid analysis of nucleic acid modification reactions in a high-throughput manner. This system allows both sensitive and nonradioactive assays to be developed for a variety of nucleic acid modification reactions. Examples presented here include assays for telomerase, uracil DNA glycosylase, polynucleotide kinase, T4 DNA ligase, C5-DNA methyltransferases, and the mismatch endonuclease CEL I. However, this approach is not confined to these reactions. Indeed the ability to perform a variety of nonradioactive assays with throughput times of 10 min per sample in conjunction with automated data analysis software represents a significant improvement in analytical and preparative nucleic acid enzymology.