The trace element selenium is found in proteins as selenocysteine (Sec), the 21st amino acid to participate in ribosome-mediated translation. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNA Sec . Its biosynthesis from seryl-tRNA Sec has been established for bacteria, but the mechanism of conversion from Ser-tRNA Sec remained unresolved for archaea and eukarya. Here, we provide evidence for a different route present in these domains of life that requires the tRNA Sec -dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA Sec kinase (PSTK) converts Ser-tRNA Sec to SeptRNA Sec . This misacylated tRNA is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS); this protein was previously annotated as SLA/LP. The human and archaeal SepSecS genes complement in vivo an Escherichia coli Sec synthase (SelA) deletion strain. Furthermore, purified recombinant SepSecS converts SeptRNA Sec into Sec-tRNA Sec in vitro in the presence of sodium selenite and purified recombinant E. coli selenophosphate synthetase (SelD). Phylogenetic arguments suggest that Sec decoding was present in the last universal common ancestor. SepSecS and PSTK coevolved with the archaeal and eukaryotic lineages, but the history of PSTK is marked by several horizontal gene transfer events, including transfer to non-Sec-decoding Cyanobacteria and fungi.aminoacyl-tRNA ͉ evolution ͉ formate dehydrogenase ͉ pyridoxal phosphate

Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was ϳ5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. In primary human infection, Epstein-Barr virus (EBV) replicates in the oropharyngeal epithelium (87) and then establishes a latent infection in B lymphocytes, which are largely nonpermissive for virus replication (68, 99). In latently infected B lymphocytes, EBV initially expresses a latency III (LTIII) program, which includes six nuclear proteins (EBNA LP, 2, 3A, 3B, 3C, and 1), two integral membrane proteins (LMP1 and LMP2), two small RNAs, EBERs, and BamA rightward transcripts (BARTs) (for a review, see references 53 and 77). EBV LTIII proteins cause infected B-lymphocyte proliferation and migration of infected B lymphocytes into lymphoid tissues. Most EBV LTIII proteins have epitopes that are recognized in the context of common major histocompatibility complex class I or II proteins and engender vigorous CD4 or CD8 T-cell responses. T-cell destruction of LTIII-infected B lymphocytes leaves some infected B lymphocytes in which LTIII gene expression has been down-regulated to LTI or LTII (42). In LTI, EBV expresses only EBNA1, EBERs, and BARTs, whereas in LTII, EBV also expresses LMP1 and LMP2. Some cells, in vivo, at least transiently express LTIII (8,102,103), since T-cell responses to LTIII-specific nuclear proteins persist throughout life.EBV replication in latently infected B lymphocytes is essential for persistent oropharyngeal replication. Prolonged acyclovir treatment effectively inhibits EBV production in the oropharynx. However, latent B-lymphocyte infection is unaffected, and EBV replication rapidly ensues when acyclovir treatment is stopped (105). Furthermore, genetically deficient humans, with X-linked agammaglobulinemia, lack mature B lymphocytes and do not have latent EBV infection in B lymphocytes or persistent oropharyngeal EBV replication (31,53,77). Since oropharyngeal EBV is essential for EBV transmission to uninfected people, EBV replication in latently infected B lymphocytes has a key role in EBV epidemiology and persistence in human populations. Also, Southern...

An innovative analytical/computational approach is presented to provide maximum allowed probabilities (MAPs) of conformations in protein domains not rigidly connected. The approach is applied to calmodulin and to its adduct with alpha-synuclein. Calmodulin is a protein constituted by two rigid domains, each of them composed by two calcium-binding EF-hand motifs, which in solution are largely free to move with respect to one another. We used the N60D mutant of calmodulin, which had been engineered to selectively bind a paramagnetic lanthanide ion to only one of its four calcium binding sites, specifically in the second EF-hand motif of the N-terminal domain. In this way, pseudocontact shifts (pcs's) and self-orientation residual dipolar couplings (rdc's) measured on the C-terminal domain provide information on its relative mobility with respect to the domain hosting the paramagnetic center. Available NMR data for terbium(III) and thulium(III) calmodulin were supplemented with additional data for dysprosium(III), analogous data were generated for the alpha-synuclein adduct, and the conformations with the largest MAPs were obtained for both systems. The MAP analysis for calmodulin provides further information on the variety of conformations experienced by the system. Such variety is somewhat reduced in the calmodulin-alpha-synuclein adduct, which however still retains high flexibility. The flexibility of the calmodulin-alpha-synuclein adduct is an unexpected result of this research.

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.