Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interaction also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

During recent studies of ribonucleolytic “degradosome” complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3′ terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3′ end of ssrA RNA.

Anti-idiotype RNA selected with an anti-nuclear export signal antibody is actively transported in oocytes and inhibits Rev- and cap-dependent RNA export

The anti-idiotype approach is based on the assumption that an antibody specific for a receptor-binding domain of a ligand could be structurally related to the receptor. Therefore, a structural mimic of a receptor-binding domain, selected with an anti-ligand antibody, might be a functional substrate for the receptor. This hypothesis was addressed here by generating antibodies recognizing the Rev-nuclear export signal (NES). A functional NES is required for active export, presumably by interacting directly or indirectly with the nuclear pore complex. Anti-NES antibodies were used to isolate RNA mimics of the NES peptide from combinatorial RNA libraries. The RNA-mimics are exported actively, block Rev-dependent export of a reporter RNA, and inhibit cap-dependent U1 snRNA export in Xenopus oocytes, properties previously reported for NES-peptide conjugates.

Numbers and Organization of RNA Polymerases, Nascent Transcripts, and Transcription Units in HeLa Nuclei

Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15,000 transcripts were elongated by polymerase I, and ∼75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in ∼2400 sites with diameters of ∼80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a “cloud” of loops around a site; each site, or transcription “factory,” would contain ∼30 active polymerases and associated transcripts.

UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged β-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

Effect of temazepam on oxygen saturation and sleep quality at high altitude: randomised placebo controlled crossover trial

Objective: To determine the effects of temazepam on the quality of sleep and on oxygen saturation during sleep in subjects at high altitude.

Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA

Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3′ major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Versatile 5′ phosphoryl coupling of small and large molecules to an RNA

A Ca2+-requiring catalytic RNA is shown to create 5′ phosphate–phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5′-diphosphates, and nonnucleotide molecules like Nɛ-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5′ phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.