Cloverleaf-like Structure Research Articles

NMR assignment of non-modified tRNAIle from Escherichia coli

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

The structural characteristics and the substrate recognition properties of RNase ZS1

TMS5 encodes an RNase ZS1 protein that can process ubiquitin-60S ribosomal protein L40 family (UbL40) mRNAs to regulate thermo-sensitive genic male sterility in rice. Despite the importance of this protein, the structural characteristics and substrate recognition properties of RNase ZS1 remain unclear. Here, we found that the variations in several conservative amino acids alter the activation of RNase ZS1, and its recognition of RNA substrates depends on the structure of RNA. RNase ZS1 acts as a homodimer. The conserved amino acids in or adjacent to enzyme center play a critical role in the enzyme activity of RNase ZS1 and the conserved amino acids that far from active center have little impact on its enzyme activity. The cleavage efficiency of RNase ZS1 for pre-tRNA-MetCAU35 and UbL401 mRNA with cloverleaf-like structure was higher than that of pre-tRNA-AspAUC9 and UbL404 mRNA with imperfect cloverleaf-like structure. This difference implies that the enzyme activity of RNase ZS1 depends on the cloverleaf-like structure of the RNA. Furthermore, the RNase ZS1 activity was not inhibited by the 5′ leader sequence and 3’ CCA motif of pre-tRNA. These findings provide new insights for studying the cleavage characteristics and substrate recognition properties of RNase ZS.

Structure basis of the FERM domain of kindlin-3 in supporting integrin αIIbβ3 activation in platelets

AbstractKindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain–containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbβ3 signaling in platelets by binding to the integrin β3 cytoplasmic tail. However, the structural details of kindlin-3's FERM domain remain unknown. In this study, we crystalized the kindlin-3's FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3's FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbβ3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbβ3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbβ3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbβ3 activation.

Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli

Transfer RNAs (tRNAs) are the most abundant class in small non-coding RNAs which have been proved to be pharmacologically active. In the present study, we evaluated the potential anticancer activities of tRNAs from Escherichia coli MRE 600 to investigate the relationship between non-pathogenic Escherichia coli strain and colorectal cancer. To purify individual tRNAs, we firstly developed a two-dimensional liquid chromatography (2D-LC) and successfully obtained two pure tRNAs. Nuclease mediated base-specific digestions coupled with UHPLC–MS/MS techniques led to an identification of these two tRNAs as tRNA-Val(UAC) and tRNA-Leu(CAG) with typical cloverleaf-like secondary structure. MTT assay demonstrated that both tRNA-1 and tRNA-2 exhibit strong cytotoxicity with IC50 of 113.0 nM and 124.8 nM on HCT-8 cells in a dose-dependent manner. Further clonogenic assay revealed that the purified tRNAs exhibit significant inhibition in colony formation with survival percentage of 79.0 ± 1.6 and 71.2 ± 2.2 at the concentration of 100 nM. These findings provided evidences of anticancer activities of tRNAs from non-pathogenic Escherichia coli strain, indicating that the pharmacological effects of these neglected biomacromolecules from microorganisms should be emphasized. This study put new insights into the therapeutic effects of intestinal microorganism on human diseases, therefore broadened our knowledge of the biological functions of gut microbiota.

Trefoil factors — new markers of gastrointestinal mucosal barrier

Trefoil factors (TFFs) are peptides containing a cloverleaf-like structure, which are synthesized in diverse organs including various regions of the human brain (presumably involved in nervous system development and differentiation), thyroid gland, mammary gland, uterus, prostate gland, conjunctiva, respiratory tract, salivary glands, and gall bladder, which peaks in diverse gastrointestinal tract mucosa: TFF-1 in the stomach, TFF-2 in the stomach and duodenum, and TFF-3 in the gut. An increasing body of studies indicate not only about a widely distributed trefoil factors in vivo but also points at their important regulatory functions. In particular, TFFs affect cell adhesion by enhancing epithelial cell migration. Recovery of reversibly damaged epithelial structures called restitution is also facilitated due to TFF-related antiapoptotic effect (anoikis resistance). In contrast, TFF-mediated proangiogenic effects can promote tumor angiogenesis, whereas their immunomodulatory effects include an influence on expression of pro-inflammatory and defense factors (including nitric oxide, cytokines, and defensins). Moreover, TFFs interacting with mucin may increase mucus viscosity, thereby protecting the mucosal layers against ulcerogenic agents. However, bronchial asthma maybe aggravated by elevating mucus viscosity in the respiratory tract due to TFF-2. In addition, TFF expression level is associated with pathogenesis of inflammatory diseases in the gastrointestinal tract. It was shown that modality of changes in TFF level might differ depending on anatomical location and severity of lesions. Changing TFF level plays an important role in oncogenesis. For instance, gastric and colorectal cancer is accompanied by upregulated TFF-1 expression. Importantly, TFF amount is considered as a diagnostic predictor due to being associated with carcinogenesis stage, metastasis as well as sensitivity to chemotherapy in gastrointestinal cancer. In addition, a role potentially played by TFFs in other malignancies including retinoblastoma, breast cancer, and thyroid carcinoma has been extensively examined. Thus, an expanding range of experimental and clinical data evidence that trefoil factors maybe considered as a promising marker of gastrointestinal and oncology diseases.

Polypyrimidine Tract-Binding Protein Regulates Enterovirus 71 Translation Through Interaction with the Internal Ribosomal Entry Site

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has caused periodic infection outbreaks in children in the Asia–Pacific region. In order to describe the largely unknown life cycle of EV71, the molecular basis of its virus-host interactions must first be determined. The 5′ untranslated region of EV71 contains a cloverleaf-like structure and internal ribosomal entry site (IRES), which play an important role in transcription and translation of viral protein. We found that polypyrimidine tract-binding protein 1 (PTB) bound to the IRES of EV71. RNA recognition motifs 1 and 2 of PTB were responsible for its binding to the EV71 IRES. Moreover, PTB protein was shuttled from nucleus to cytoplasm after EV71 infection. Additionally, IRES activity and viral protein production were inhibited by PTB knockdown. These results suggest that PTB interacts with the EV71 IRES, and positively regulates viral protein translation.

The mitochondrial genome of the oribatid mite Paraleius leontonychus: new insights into tRNA evolution and phylogenetic relationships in acariform mites

Bilaterian mitochondrial (mt) genomes are circular molecules that typically contain 37 genes. To date, only a single complete mitogenome sequence is available for the species-rich sarcoptiform mite order Oribatida. We sequenced the mitogenome of Paraleius leontonychus, another species of this suborder. It is 14,186 bp long and contains 35 genes, including only 20 tRNAs, lacking tRNAGly and tRNATyr. Re-annotation of the mitogenome of Steganacarus magnus increased the number of mt tRNAs for this species to 12. As typical for acariform mites, many tRNAs are highly truncated in both oribatid species. The total number of tRNAs and the number of tRNAs with a complete cloverleaf-like structure in P. leontonychus, however, clearly exceeds the numbers previously reported for Sarcoptiformes. This indicates, contrary to what has been previously assumed, that reduction of tRNAs is not a general characteristic for sarcoptiform mites. Compared to other Sarcoptiformes, the two oribatid species have the least rearranged mt genome with respect to the pattern observed in Limulus polyphemus, a basal arachnid species. Phylogenetic analysis of the newly sequenced mt genome and previously published data on other acariform mites confirms paraphyly of the Oribatida and an origin of the Astigmata within the Oribatida.

Novel Genomic and Evolutionary Perspective of Cyanobacterial tRNAs.

Transfer RNA (tRNA) plays a central role in protein synthesis and acts as an adaptor molecule between an mRNA and an amino acid. A tRNA has an L-shaped clover leaf-like structure and contains an acceptor arm, D-arm, D-loop, anti-codon arm, anti-codon loop, variable loop, Ψ-arm and Ψ-loop. All of these arms and loops are important in protein translation. Here, we aimed to delineate the genomic architecture of these arms and loops in cyanobacterial tRNA. Studies from tRNA sequences from 61 cyanobacterial species showed that, except for few tRNAs (tRNAAsn, tRNALeu, tRNAGln, and tRNAMet), all contained a G nucleotide at the 1st position in the acceptor arm. tRNALeu and tRNAMet did not contain any conserved nucleotides at the 1st position whereas tRNAAsn and tRNAGln contained a conserved U1 nucleotide. In several tRNA families, the variable region also contained conserved nucleotides. Except for tRNAMet and tRNAGlu, all other tRNAs contained a conserved A nucleotide at the 1st position in the D-loop. The Ψ-loop contained a conserved U1-U2-C3-x-A5-x-U7 sequence, except for tRNAGly, tRNAAla, tRNAVal, tRNAPhe, tRNAThr, and tRNAGln in which the U7 nucleotide was not conserved. However, in tRNAAsp, the U7 nucleotide was substituted with a C7 nucleotide. Additionally, tRNAArg, tRNAGly, and tRNALys of cyanobacteria contained a group I intron within the anti-codon loop region. Maximum composite likelihood study on the transition/transversion of cyanobacterial tRNA revealed that the rate of transition was higher than the rate of transversion. An evolutionary tree was constructed to understand the evolution of cyanobacterial tRNA and analyses revealed that cyanobacterial tRNA may have evolved polyphyletically with high rate of gene loss.

MOV10 interacts with Enterovirus 71 genomic 5′UTR and modulates viral replication

As a cytoplasmic parasite, RNA virus develops sophisticated mechanisms to counter host defense and utilize host proteins to facilitate its replication. Here we found Moloney leukemia virus 10 (MOV10), a highly conserved cellular protein belonging to SF1 helicase family, played critical roles in EV71 infection. Silencing cellular MOV10 could restrict EV71 replication, while over-expressing MOV10 resulted in increased viral replication at low dosage and repressed viral replication at high dosage. Further investigation showed that MOV10 exhibited dual functions in EV71 regulation, its C-terminus positively regulated viral replication by binding to EV71 cloverleaf-like structure and the internal ribosome entry site while the N-terminus showed a potential antiviral activity when individually overexpressed. In addition, RNA-dependent interaction between MOV10 and HuR as well as the co-localization of MOV10 and processing bodies were also observed post infection. Taken together, our data indicate a crucial role of MOV10 in EV71 infection for the first time, providing new insights for its roles in EV71 infection.

Domain I of the 5′ non-translated genomic region in coxsackievirus B3 RNA is not required for productive replication

Domain I is a cloverleaf-like secondary structure at the 5′ termini of all enterovirus genomes, comprising part of a cis-acting replication element essential for efficient enteroviral replication. 5′ genomic terminal deletions up to as much as 55% of domain I can occur without lethality following coxsackie B virus infections. We report here that the entire CVB structural domain I can be deleted without lethality.

Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA.

The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200 nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1908-0) contains supplementary material, which is available to authorized users.

Biological evidence for the world's smallest tRNAs

Due to their function as adapters in translation, tRNA molecules share a common structural organization in all kingdoms and organelles with ribosomal protein biosynthesis. A typical tRNA has a cloverleaf-like secondary structure, consisting of acceptor stem, D-arm, anticodon arm, a variable region, and T-arm, with an average length of 73 nucleotides. In several mitochondrial genomes, however, tRNA genes encode transcripts that show a considerable deviation of this standard, having reduced D- or T-arms or even completely lack one of these elements, resulting in tRNAs as small as 66nts. An extreme case of such truncations is found in the mitochondria of Enoplea. Here, several tRNA genes are annotated that lack both the D- and the T-arm, suggesting even shorter transcripts with a length of only 42nts. However, direct evidence for these exceptional tRNAs, which were predicted by purely computational means, has been lacking so far. Here, we demonstrate that several of these miniaturized armless tRNAs consisting only of acceptor- and anticodon-arms are indeed transcribed and correctly processed by non-encoded CCA addition in the mermithid Romanomermis culicivorax. This is the first direct evidence for the existence and functionality of the smallest tRNAs ever identified so far. It opens new possibilities towards exploration/assessment of minimal structural motifs defining a functional tRNA and their evolution.

Tumor Antigen Small Guide (sg) RNA as a Completely Unique Nucleic Acid Medicine for Leukemia and Other Tumors.

Abstract 2461Poster Board II-4383′ trailers of pre-tRNA or micro-pre-tRNA are removed by tRNA 3′ processing endoribonuclease (tRNase ZL) for pre-tRNA or micro-pre-tRNA to transform into mature tRNA or micro-tRNA. It has been demonstrated that 7-base-pair acceptor stem, about 5-base pair T-loop structure and a 3′-trailer are requisites for being recognized by tRNase ZL. A synthesized small guide RNA (sgRNA), which is designed to possess the complementary sequences with target RNA and form sgRNA/target RNA complex resembling a pre-tRNA or a micro-pre-tRNA, could be used for target RNA to be recognized and cleaved. As to the structure of sgRNA, a 3′-truncated tRNA (a clover leaf-like structure resembling tRNA and complementary to target RNA in the 7-base-pair acceptor stem) and a 5′-half-tRNA (a half of clover leaf-like structure and complemantary to target RNA of a 3′-half-tRNA structure) can form a pre-tRNA-like complex with target RNA. Heptamer sgRNA (7-base RNA structure complementary to target RNA and making target RNA form T-loop structur downstream of the complementary region) as well as hook RNA (a single T-loop-like structure with upstream 7-base RNA structure complementary to target RNA) can form a micro-pre-tRNA-like complex, which could be recognized and cleaved by tRNase Z. Heptamer sgRNA is thought to be superior to siRNA in mucleic acid medicine because of less off-target effect, less IFN-α production, easier introduction into target cells and less expensive for synthesis. In order to develop a unique sgRNA-based nucleic acid medicine, we investigated whether apoptosis could sbe induced and proliferation could be inhibited in leukemia cells by using tumor antigen heptamer sgRNA. The 5′, 3′-phosphorylated 2′-O-methyl sgRNAs with or without 3′LNA (locked nucleic acid), which were designed to form micro-pre-tRNA like structure with target mRNA of leukemia-associated antigens (LAA) such as WT1, Bcl2, PRAME, survivin, proteinase 3, hTERT and RHAMM, were synthesized and purified by high-performance liquid chromatography. Several leukemic cell lines and fresh leukemia cells from AML/ALL patients expressing relevant LAA were cultured with each heptamer sgRNA at the concecntrations of 0.1 μM to 10 μM for 24-72 hours. Effecs of sgRNA on apoptosis of the leukemia cells were evaluated by flow cytometry after staining with Annexin-V/7AAD or DiOC6(3). Proliferaton inhibition of the cells by sgRNA was evaluated by an improved MTT assay (TetraColor One). In addition, introduction of heptamer sgRNA into target cells was investigated using FITC-labeled sgRNA. Although EGFP sgRNA (negative control) did not induce apoptosis in leukemia cells such as K562, heptamer sgRNA against WT1, Bcl2, PRAME, surviving, proteinase 3, hTERT and RHAMM could induce apoptosis in concentration dependent manner of sgRNA irrespective of sgRNA with or without LNA. Apoptosis was enhanced by the combination of two different sgRNAs such as WT1 sgRNA and PRAME sgRNA. Likewise, proliferation was inhibited in leukemia cells cultured with sgRNA against WT1, PRAME, survivin, proteinase 3, hTERT and RHAMM. Apoptosis due to anti-leukemic agent such as etoposide or doxorubicine was enhanced by the addition of sgRNA more than the additive theoretical level of anti-leukemic agent and sgRNA. Apoptosis of some leukemia cells such as HL60, which were resistant to LAA sgRNA in apoptosis or proliferation inhibition, could be hardly introduced into with FITC-labeled sgRNA by only culturing with. sgRNA-based apoptosis in these cell lines such as HL60 was enhanced by using lipofection for sgRNA intruduction. These findings suggested a possibility that tumor antigen sgRNA could be applied as a novel nucleic acid medicine against leukemia and other tumors. Disclosures:No relevant conflicts of interest to declare.

Evidence for Variable Selective Pressures at a Large Secondary Structure of the Human Mitochondrial DNA Control Region

A combined effect of functional constraints and random mutational events is responsible for the sequence evolution of the human mitochondrial DNA (mtDNA) control region. Most studies targeting this noncoding segment usually focus on its primary sequence information disregarding other informative levels such as secondary or tertiary DNA conformations. In this work, we combined the most recent developments in DNA folding calculations with a phylogenetic comparative approach in order to investigate the formation of intrastrand secondary structures in the human mtDNA control region. Our most striking results are those regarding a new cloverleaf-like secondary structure predicted for a 93-bp stretch of the control region 5'-peripheral domain. Randomized sequences indicated that this structure has a more negative folding energy than the average of random sequences with the same nucleotide composition. In addition, a sliding window scan across the complete mitochondrial genome revealed that it stands out as having one of the highest folding potential. Moreover, we detected several lines of evidence of both negative and positive selection on this structure with high levels of conservation at the structure-relevant stem regions and the occurrence of compensatory base changes in the primate lineage. In the light of previous data, we discuss the possible involvement of this structure in mtDNA replication and/or transcription. We conclude that maintenance of this structure is responsible for the observed heterogeneity in the rate of substitution among sites in part of the human hypervariable region I and that it is a hot spot for the 3' end of human mtDNA deletions.

Distinctive structures between chimpanzee and humanin a brain noncoding RNA

Human accelerated region 1 (HAR1) is a short DNA region identified recently to have evolved the most rapidly among highly constrained regions since the divergence from our common ancestor with chimpanzee. It is transcribed as part of a noncoding RNA specifically expressed in the developing human neocortex. Employing a panoply of enzymatic and chemical probes, our analysis of HAR1 RNA proposed a secondary structure model differing from that published. Most surprisingly, we discovered that the substitutions between the chimpanzee and human sequences led the human HAR1 RNA to adopt a cloverleaf-like structure instead of an extended and unstable hairpin in the chimpanzee sequence. Thus, the rapid evolutionary changes resulted in a profound rearrangement of HAR1 RNA structure. Altogether, our results provide a structural context for elucidating HAR1 RNA function.

Assembly of a two-dimensional oxalate-bridged heterometallic CoII3CrIII2 coordination polymer

Reaction of CoCl(2).4H(2)O with Ag(3)[Cr(ox)(3)] (3 : 2 mol/mol) in methanol gives rise to an oxalate-bridged complex [Co(MeOH)(2)](3)[Cr(ox)(3)](2).6MeOH that consists of a rare cloverleaf-like layered structure (12,3-net). Ferromagnetic coupling is operative between the adjacent metal ions through the oxalate bridges.

Crystal Structure of Human T-protein of Glycine Cleavage System at 2.0 Å Resolution and its Implication for Understanding Non-ketotic Hyperglycinemia

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.

Functional analysis of the cloverleaf-like structure in the 3′ untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement

The 3′ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/ΔABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/ΔA, -/ΔB, and -/ΔC each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40A/ΔB and -/ΔC were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMV/ΔA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain.

Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity

In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S1 mapping we defined the 3′-end at 8423(±1); thus CI RNA is 79(±1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5′-end and the CCA sequence at the 3′-end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.