Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition.

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."     

Tails of RNA polymerase II

Eukaryotic RNA polymerase II contains two distinct structural domains: a catalytic core consisting of subunits that are homologous to other multisubunit RNA polymerases, and a unique extension of the carboxy-terminus of the largest subunit comprising tandem repeats of the seven amino acid sequence YSPTSPS. This repetitive 'tail' domain is essential for polymerase function in vivo. Although the nature of this essential function is unknown, actively transcribing RNA polymerase II is known to be multiphosphorylated on this repetitive domain.     

A cryptic RNA-binding domain in the Pol region of the L-A double-stranded RNA virus Gag-Pol fusion protein.

The Pol region of the Gag-Pol fusion protein of the L-A double-stranded (ds) RNA virus of Saccharomyces cerevisiae has (i) a domain essential for packaging viral positive strands, (ii) consensus amino acid sequence patterns typical of RNA-dependent RNA polymerases, and (iii) two single-stranded RNA binding domains. We describe here a third single-stranded RNA binding domain (Pol residues 374 to 432), which is unique in being cryptic. Its activity is revealed only after deletion of an inhibitory region C terminal to the binding domain itself. This cryptic RNA binding domain is necessary for propagation of M1 satellite dsRNA, but it is not necessary for viral particle assembly or for packaging of viral positive-strand single-stranded RNA. The cryptic RNA binding domain includes a sequence pattern common among positive-strand single-stranded RNA and dsRNA viral RNA-dependent RNA polymerases, suggesting that it has a role in RNA polymerase activity.     

Mechanism for the alteration of the substrate specificities of template-independent RNA polymerases

PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197). The pocket structure is sustained by interactions between the catalytic domain and the RNA-binding domain. EcPAP has a flexible basic C-terminal region that contributes to optimal RNA translocation for processive adenosine 5'-monophosphate (AMP) incorporations onto the 3'-end of RNAs. A comparison of the EcPAP structure with those of other template-independent RNA polymerases suggests that structural changes of domain(s) outside the conserved catalytic core domain altered the substrate specificities of the template-independent RNA polymerases.     

Evolution of RNA-dependent RNA polymerases of positive riboviruses

A comparative analysis is presented of 24 known amino acid sequences of RNA-dependent RNA polymerases of positive strand RNA viruses infecting animals, plants and bacteria. Using a newly proposed methodology of group alignment for weakly similar sequences, evolutionary conserved fragments of all these proteins were unambiguously aligned. A unique pattern (consensus) of 7 invariant amino acid residues was revealed which is absent from the sequences of other RNA and DNA polymerases and is thought to unequivocally identify the RNA-dependent RNA polymerases of positive strand RNA viruses. Based on the obtained alignment a tentative phylogenetic tree of viral RNA polymerases was constructed for the first time. The RNA-dependent RNA polymerases of positive strand RNA viruses are concluded to comprise a distinct family of evolutionary related proteins.     

Nuclear localization of senescence marker protein-30, SMP30, in cultured mouse hepatocytes and its similarity to RNA polymerase

Senescence marker protein-30 (SMP30), expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity. By immunohistochemistry and western blotting, we found that SMP30 was in both the nuclei and cytoplasm of cultured mouse hepatocytes. By a homology search, we found that a domain of the SMP30 sequence 51 amino acid residues long was 60-66% similar to bacterial and yeast RNA polymerases.     

Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.     

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.     

Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T = 9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.     

Identification of a region of the bacteriophage T3 and T7 RNA polymerases that determines promoter specificity

Bacteriophages T7 and T3 encode DNA-dependent RNA polymerases that are 82% homologous, yet exhibit a high degree of specificity for their own promoters. A region of the RNA polymerase gene (gene 1) that is responsible for this specificity has been localized using two approaches. First, the RNA polymerase genes of recombinant T7 x T3 phage that had been generated in other laboratories in studies of phage polymerase specificity were characterized by restriction enzyme mapping. This approach localized the region that determines promoter specificity to the 3' end of the polymerase gene, corresponding to the carboxyl end of the polymerase protein distal to amino acid 623. To define more closely the region of promoter specificity, a series of hybrid T7/T3 RNA polymerase genes was constructed by in vitro manipulation of the cloned genes. The specificity of the resulting hybrid RNA polymerases in vitro and in vivo indicates that an interval of the polymerase that spans amino acids 674 to 752 (the 674 to 752 interval) contains the primary determinant of promoter preference. Within this interval, the amino acid sequences of the T3 and T7 enzymes differ at only 11 out of 79 positions. It has been shown elsewhere that specific recognition of T3 and T7 promoters depends largely upon base-pairs in the region from -10 to -12. An analysis of the preference of the hybrid RNA polymerases for synthetic T7 promoter mutants indicates that the 674 to 752 interval is involved in identifying this region of the promoter, and suggests that another domain of the polymerase (which has not yet been identified) may be involved in identifying other positions where the two consensus promoter sequences differ (most notably at position -15).