Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena.

The rRNA intervening sequence of Tetrahymena is a catalytic RNA molecule, or "ribozyme." A tertiary-structure model of the active site of this ribozyme has been constructed based on comparative sequence analysis of related group I intervening sequences, data on the accessibility of each nucleotide to chemical and enzymatic probes, and principles of RNA folding derived from a consideration of the structure of tRNA determined by x-ray crystallography. In the model, the catalytic center has a two-helix structural framework composed of the base-paired segments of the group I conserved sequence elements. The structural framework supports and orients the conserved nucleotides that are adjacent to the base-paired sequence elements; these conserved nucleotides are proposed to form the active site and to bind the 5' splice-site duplex and the guanine nucleotide substrate. Tests of the model are proposed.     

Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center

Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2–3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351–363] proposed that rifamycins function by allosteric modulation of binding of Mg²⁺ to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg²⁺ to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg²⁺ to the RNAP active center.     

The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV.

The lack of small-animal models that are suitable for evaluation of agents used to treat infection with hepatitis C virus (HCV) severely hinders the assessment of potential new therapies for the disease. This study created such a model, termed the "HCV-Trimera" model. The HCV-Trimera model was developed by using lethally irradiated mice, reconstituted with SCID mouse bone marrow cells, in which human liver fragments infected ex vivo with HCV had been transplanted. Viremia (positive-strand HCV RNA levels) in HCV-Trimera mice peaked at approximately day 18 after liver transplantation, and an infection rate of 85% was reached. Viral replication in liver grafts was evidenced by the presence of specific negative-strand HCV RNA. The usefulness of this model for evaluation of anti-HCV agents was demonstrated by the ability of a small molecule (an HCV internal ribosomal entry site inhibitor) and an anti-HCV human monoclonal antibody (HCV AB(XTL)68) to reduce virus loads in HCV-Trimera mice in a dose-dependent manner.     

The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH(4)(+) or NH(3)) of the transported species. We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-A resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH(4)(+) is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH(3.) The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.     

Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process.     

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn²⁺ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.     

Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells

Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N-acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication.     

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5'-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome.     

La protein is a potent regulator of replication of hepatitis C virus in patients with chronic hepatitis C through internal ribosomal entry site-directed translation

BACKGROUND AND AIMS: Translation of hepatitis C virus is an essential step of viral replication and is mediated by an internal ribosome entry site. We previously reported that the hepatitis C virus internal ribosome entry site is most active during the synthetic (S) or mitotic (M) phases and lowest during quiescent (G 0 ) phase. Here, we investigated host factors responsible for the regulation of the hepatitis C virus internal ribosome entry site. METHODS: We synchronized the cell-cycle progression and evaluated gene-expression dynamics of host factors and kinetics of hepatitis C virus internal ribosome entry site activity in cells at various points during the cell cycle by using a complementary DNA microarray. We also validated the significance of identified host factors on hepatitis C virus replication in vivo. RESULTS: Hepatitis C virus internal ribosome entry site activity correlated with a gene cluster induced in the S and G 2 /M phases. It is interesting to note that most initiation factors known to bind or interact with the hepatitis C virus internal ribosome entry site [poly(rC)-binding protein 2, polypyrimidine tract binding protein, eukaryotic initiation factor 3, eukaryotic initiation factor 2gamma, eukaryotic initiation factor 2beta, La protein, and heterogenous nuclear ribonucleoprotein L] were induced during the S and G 2 /M phases. Expression of La protein, polypyrimidine tract binding protein, and eukaryotic initiation factor 3 (p116, p170) were predominantly repressed in G 0 phase and induced in S and G 2 /M phases. Suppression or overexpression of La protein and polypyrimidine tract binding protein in RCF-26 significantly changed hepatitis C virus internal ribosome entry site activity. In the livers of patients with chronic hepatitis C, expression of La protein was significantly increased and correlated with the amount of hepatitis C virus RNA. CONCLUSIONS: Hepatitis C virus uses host factors induced during cell division but not during quiescence for replication. Of these, La protein is a potent regulator and enhances hepatitis C virus replication in regenerating hepatocytes in patients with chronic hepatitis C.     

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

Chitinase B (ChiB) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites -2 to +3) shows closure of the "roof" of the active site tunnel. It also shows that the sugar in the -1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.     

The role of the allosteric B site in the fumarase reaction

The role of a malate binding site in a concavity external to the more deeply situated active site has been a major mystery of the fumarase reaction. The malate, within 12 A of the active site, was bound by hydrogen bonds to two main-chain amides and to two basic residues, H129 and R126. Mutation of the His of this so-called B site of Escherichia coli fumarase had little effect on the overall initial rate kinetics of the enzyme, which has obscured an understanding of the critical role of the site. Contrary to the WT enzyme, which is rate-limited in the recycling of free enzyme isoforms that follows product release, the enzyme with both basic residues modified is rate-limited in the product release step itself. A loss of complexity in the mutated, but still functional, step is indicated by a greatly reduced sensitivity of its rate to changes in temperature. Unlike the inhibition by glycerol shown with normal enzyme and attributed to a viscogenic effect on the recycling rate, the product-release step of the B-site mutants is accelerated by glycerol, suggestive of a structural effect on the 12-A space between the A and B sites. It is proposed that the "extra" malate represents a stage in the transfer of substrate and product between the solvent and the "buried" active site of the enzyme.