The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

No abstract

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RP021). A screen for mutations in RP021 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo2l mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo2l mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo2l mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.Eukaryotic RNA polymerases (RNAPs) are highly conserved, multisubunit enzymes composed of two large subunits associated with several smaller polypeptides, some of which are shared by all three RNAPs or are common to only RNAPI and RNAPIII (reviewed in references 68, 73, and 83). In addition to RNAPII, mRNA synthesis requires several general factors for the assembly of initiation complexes at promoters and subsequently the stimulation of transcript elongation and termination (reviewed in references 66 and 68). Although gene expression is most often regulated at the level of transcription initiation in eukaryotes, a growing number of studies indicate that control of gene expression also occurs at the level of transcript elongation (reviewed in references 37 and 79). In eukaryotes and their viruses, examples of genes known to be regulated at the level of transcript elongation include the human immunodeficiency virus type 1 long terminal repeat (60), the simian virus 40 late-transcription unit (4), the major late-transcription unit of adenovirus (44), the proto-oncogenes c-myc (8), c-myb (6), c-mos (48), c-fos (15, 49), and L-myc (41), as well as the adenosine deaminase gene (13). In Drosophila melanogaster, work performed by Gilmour and Lis (26) identified, little is known about the factors that may be involved in this process. Clear...

The DNA sequences of eight yeast ribosomal protein genes have been compared for the purpose of identifying homologous regions which may be involved in the coordinate regulation of ribosomal protein synthesis. A 12 bp homology was identified in the 5' DNA sequence preceding the structural gene for 6 out of 8 yeast ribosomal protein genes. In each case the homologous sequence was found at a position approximately 300 bp preceding the transcription start of the ribosomal protein gene. This homology was not identified in any non-ribosomal protein gene examined. Additional homologies between ribosomal protein genes were identified in the transcribed regions, including the untranslated 5' and 3' DNA regions flanking the coding regions.

We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxinconverting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted Al fragment of the SLT-1 A subunit. SI nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene op plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Shigella dysenteriae 1 produces a toxin which is cytotoxic to eucaryotic cell lines (8). Binding to the glycolipid globotriaosylceramide (Gb3) membrane receptor is mediated by a pentamer of 7-kilodalton (kDa) B subunits, while the 31-kDa A subunit, after proteolytic nicking and reduction, inhibits protein synthesis by catalytic inactivation of the 60S ribosomal subunit (8,20,34,35). O'Brien and LaVeck (30,31) have shown that some Escherichia coli strains produce large amounts of a cytotoxin which appeared very similar to Shiga toxin with respect to its subunit structure and mechanism of action. It was completely neutralized by antiserum raised against Shiga toxin and was named Shiga-like toxin 1 (SLT-1) (28, 42). Recently, Strockbine et al. (42) have characterized a second cytotoxin, also produced by E. coli, which is related to SLT-1 at the DNA sequence level but is not neutralized by antiserum raised against Shiga toxin or SLT-

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.Cellular processes in which RNA plays an essential role are likely to involve proteins whose function is the unwinding of RNA-RNA (or RNA-DNA) duplexes. Until recently only a few RNA helicases have been characterized biochemically. These include the Escherichia coli transcription termination factor Rho (7), the simian virus 40 large T antigen (53), the eukaryotic translation initiation factor eIF4A (45), and the nuclear protein P68 (17), which shares similarity with both eIF4A and the simian virus 40 large T antigen. Recently a number of gene products have been identified which, on the basis of their homology with eIF4A, are presumed to be ATP-dependent RNA helicases. These gene products, which make up the so-called D-E-A-D family of proteins (34), are thought to function primarily in events that occur posttranscriptionally. This growing family includes the following: the Drosophila vasa gene product (28) and the murine spermspecific protein PL10 (32), both of which are thought to be involved in temporal or tissue-specific mRNA translation; the yeast proteins PRP5 (14) and PRP-16 (11) and yeast mitochondrial protein MSS116 (54), which participate in pre-mRNA splicing; and the yeast translation initiation factors TIF1/2 (35) and SPB4 (49) and E. coli SrmB (41), which play roles in mRNA translation and ribosome assembly, respectively.The E. coli ribosome is the prototypical ribonucleoprotein particle. As with other ribonucleoprotein particles, there is a growing recognition that at least some of the catalytic activity associated with the ribosome resides in ligandinduced conformational changes in its RNA component, with the ribosomal proteins serving as modulators of the RNA structure. Translation of mRNA in E. coli requires not only ribosomes, consisting of 53 ribosomal proteins and 3 rRNAs, but also tRNAs, aminoacyl-tRNA synthetases, and tRNA-modifying enzymes. In addition, translation requires proteins transiently associated with the ribosome, including initiation factors (IF-1, IF-2, and IF-3), elongation factors (EF-G, EF-Tu, and EF-Ts), and release factors (RF-1, RF-2, and RF-3 [23]). One would assume that the repertoire of * Corresponding author.proteins necessary for translation in vivo might include the proteins whose function is the unwinding of RNA duplexes. These might facilitate conformational changes in rRNA, tRNA-rRNA interactions, and the melting of mRNA secondary structure. One D-...

Summary. Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1␤, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype.