NTP-binding Protein Research Articles

Genetic studies on the poliovirus 2C protein, an NTPase A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization

Poliovirus RNA replication is known to be inhibited by millimolar concentrations of guanidine. A variety of guanidine-resistant ( gr) and guanidine-dependent ( gd) poliovirus strains were selected, and mutations responsible for the phenotypic alterations were mapped to distinct loci of the viral NTP-binding pattern containing protein 2C. Together with already published results, our data have demonstrated that the overwhelming majority of guanidine mutants of poliovirus 2C can be assigned to one of the two classes, N (with a change in Asn179) or M (with a change in Met187). As inferred from the structure/function relations in other NTP-binding proteins, both these “main” mutations should reside in a loop adjoining the so-called B motif known to interact with the Mg 2+, involved in the NTP splitting. In classes M (always) and N (not infrequently), these B motif mutations were combined with mutations in, or close to, motif A (involved in binding of the NTP phosphate moieties) and/or motif C (another conserved element of a subset of NTP-binding proteins). These data strongly support the notion that the region of polypeptide 2C involved in the NTP utilization is affected by the guanidine mutations and by the presence of the drug itself. The mutations, however, never altered highly conserved amino acid residues assumed to be essential for the NTP binding or splitting. These facts and some other considerations led us to propose that guanidine affects coupling between the NTP binding and/or splitting, on the one hand, and the 2C function (related to conformational changes), on the other. Both N and M classes of mutants contain gr and gd variants, and the gr/gd interconversion as well as modulations of the guanidine phenotype can be caused by additional mutations within each class; sometimes, these additional substitutions are located far away from the “main” mutations. It is suggested that the target for guanidine action involves long-range tertiary interactions. Under conditions restrictive for the individual growth of each parent, efficient reciprocal intra-allelic complementation between guanidine-sensitive ( gs) and gd strains (of M or N classes) was observed. The complementation occurred at the level of viral RNA synthesis. These data allowed us to propose that oligomerization of polypeptide 2C is an essential step in the replication of viral genome.

Nucleotide Sequence and Genomic Organization of Rice Tungro Spherical Virus

Rice tungro disease is caused by a combination of two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (Jones et al. (1991) J. Gen. Virol. 72, 757-761.). The genome of RTSV is a single-stranded polyadenylated RNA. We present here the 12,433-nucleotide complete sequence of RTSV genomic RNA and its deduced coding regions. This sequence contains a large open reading frame (ORF) which initiates following a 514-nucleotide 5′ leader sequence and is capable of encoding a viral polyprotein of 390.3 kDa. Two viral subgenomic RNAs of ca. 1.2 and 1.4 kb, respectively, were detected in RTSV-infected leaf tissues and mapped by S1 nuclease protection assay. These RNAs were determined to be congruent with the genomic RNA sequence proximal to the 3′ terminus and could contain up to two small ORFs in their 5′ to 3′ orientation. There are at least three capsid protein subunit cistrons near the N-terminus of the large ORF. A computer-aided search of the C-terminal half of the large ORF revealed conserved protein sequence motifs for a viral RNA polymerase, proteinase, and a putative NTP-binding protein. These sequence motifs are arranged in a manner that resembles those of picorna-like viruses. Taken together, these data indicate that RTSV is a distinct type of positive-strand RNA virus. The evolutionary relationships between RTSV and other picorna-like plant viruses are discussed.

ATP-GTP(NTP)-Binding Proteins and Light Signal Transmission in the Plasma Membrane from Etiolated Pea Seedlings.

NTP-binding proteins were detected in the plasma membrane purified from elongating third internodes of etiolated pea (Pisum sativum Alaska) seedlings. After [α-32P] ATP and [α-32P] GTP bound to proteins in the plasma membrane, the reaction mixtureas were UV crosslinked, then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Pretreatment of the membrane fraction by a Ras-specific antibody efficiently blocked the binding of [α-32P] ATP and [α-32P] GTP. Several proteins in the plasma membrane were [32P] ADP-ribosylated by endogenous ADP-ribosyl transferase, and by cholera and pertussis toxins. The [32P] ADP-ribosylation of these proteins by endogenous ADP-ribosyl transferase was partly reduced by pretreating the plasma membrane with a Ras-specific antibody. Most of the [α-32P]ATP binding to several NTP-binding proteins was inhibited efficiently by 10-3 M ATP. The binding of [α-32P] GTP was inhibited more efficiently, however, by 10-3 M ATP or UTP than by 10-3 M GTP or CTP. Red light irradiation of the third internodes stimulated the binding of [α-32P] ATP and [α-32P] GTP to 16, 21, 32, 34, 37, 71 and 92 kDa proteins, as analyzed by UV-induced crosslinking followed by SDS-PAGE, and the results were supported by using Triton X-100-PAGE without UV-induced crosslinking. Among these, the amount of a 37 kDa protein exhibiting cross-reactivity with α subunit of transducin-sepcific antibody, was rapidly reduced in the plasma membrane after red light irradiation of the third internodes. A 30 kDa protein showing cross reactivity with β, γ subunit-specific antibody, however, stayed relatively unchanged.

Sequence of the 3? half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5

We have determined the nucleotide sequence of the 3'-terminal half of the RNA genome of Murray Valley encephalitis virus (MVE) using seven overlapping cDNA clones; an estimated 80-90 nucleotides at the extreme 3'-end remain to be sequenced. In conjunction with previous sequence data for the 5' half (16), we can conclude that the MVE genome contains a long open reading frame of 10,302 nucleotides that encodes a polyprotein of 3434 residues. Comparison of the MVE deduced amino acid sequence with that of other flaviviruses shows that MVE is most closely related to Japanese encephalitis virus, consistent with serological studies. Using N-terminal amino acid sequence analysis, three nonstructural proteins (NS1, NS3, and NS5) have been identified and mapped on the MVE genome. MVE NS3 contains sequence motifs suggesting that its amino terminus may function as a serine protease. The central region of NS3 (in the linear amino acid sequence) has motifs that are found in NTP-binding proteins and helicases. MVE NS5 contains a conserved Gly-Asp-Asp sequence that is thought to be essential for RNA-dependent RNA polymerases.

Superfamily of UvrA-related NTP-binding proteins: Implications for rational classification of recombination/repair systems

A superfamily of proteins encoded by bacterial, phage and eukaryotic genomes and performing a wide range of NTP-dependent functions was delineated by amino acid sequence comparison. The new superfamily brought together bacterial proteins UvrA, RecF, RecN, MutH and HexA, T4 phage gp46, T5 phage D13 protein, lambda phage EA59 protein and yeast Rad50 protein, all involved in recombination, repair and, in some cases, also in replication of respective genomes, and a family of bacterial and eukaryotic proteins implicated in active transport of various compounds, cell division and nodulation whose relationship to UvrA had been recognized previously. For some of the members of the new superfamily, NTPase activity or NTP-binding capacity have been demonstrated. All these proteins encompassed four distinct conserved sequence motifs, of which two constituted the NTP-binding pattern typical of a vast class of ATP and GTP-binding proteins, whereas the other two were unique for the new superfamily. The new superfamily was characterized by an unusually large span of length variation of polypeptide segments separating the two conserved motifs of the NTP-binding pattern. Sequence similarity was revealed, on the one hand, between the N-terminal NTP-binding domain of UvrA, recN, gp46 and D13, and on the other hand, between the C-terminal NTP-binding domain of UvrA, recF and EA59. Possible relationships between different pathways of DNA repair and recombination are briefly analyzed from the viewpoint of involvement of NTPases of different groups.

A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses

Statistically significant similarity was revealed between amino acid sequences of NTP-binding pattern-containing domains which are among the most conserved protein segments in dissimilar groups of ss and dsDNA viruses (papova-, parvo-, geminiviruses and P4 bacteriophage), and RNA viruses (picorna-, como- and nepoviruses) with small genomes. Within the aligned domains of 100–120 amino acid residues, three highly conserved sequence segments have been identified, i.e. ‘A’ and ‘B’ motifs of the NTP-binding pattern, and a third, C-terminal motif ‘C’, not described previously. The sequence of the ‘B’ motif in the proteins of the new superfamily is unusually variable, with substitutions, in some of the members, of the Asp residue conserved in other NTP-binding proteins. The ‘C’ motif is characterized by an invariant Asn residue preceded by a stretch of hydrophobic residues. As the new superfamily included a well studied DNA and RNA helicase, T antigen of SV40, helicase function could be tentatively assigned also to the other related viral putative NTP-binding proteins. On the other hand, the possibility of different and/or multiple functions for some of these proteins is discussed.

A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing

We previously described a dominant suppressor of the splicing defect conferred by an A→;C intron branchpoint mutation in S. cerevisiae. Suppression occurs by increasing the frequency with which the mutant branchpoint is utilized. We have now cloned the genomic region encoding the prp16-1 suppressor function and have demonstrated that PRP16 is essential for viability. A 1071 amino acid open reading frame contains sequence motifs characteristic of an NTP binding fold and further similarities to a superfamily of proteins that includes members with demonstrated RNA-dependent ATPase activity. A single nucleotide change necessary to confer the prp16-1 suppressor phenotype results in a Tyr→Asp substitution near the “A site” consensus for NTP binding proteins. We propose that PRP16 is an excellent candidate for mediating one of the many ATP-requiring steps of spliceosome assembly and that accuracy of branchpoint recognition may be coupled to ATP binding and/or hydrolysis.