Transfer RNA editing in land snail mitochondria.

Some mitochondrial tRNA genes of land snails show mismatches in the acceptor stems predicted from their gene sequences. The majority of these mismatches fall in regions where the tRNA genes overlap with adjacent downstream genes. We have synthesized cDNA from four circularized tRNAs and determined the sequences of the 5' and 3' parts of their acceptor stems. Three of the four tRNAs differ from their corresponding genes at a total of 13 positions, which all fall in the 3' part of the acceptor stems as well as the discriminator bases. The editing events detected involve changes from cytidine, thymidine, and guanosine to adenosine residues, which generally restore base-pairing in the stems. However, in one case an A-A mismatch is created from an A-C mismatch. It is suggested that this form of RNA editing may involve polyadenylylation of the maturing tRNAs as an intermediate.

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem–loop structure located 15–43 nt upstream of the 5′ end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA.

DNA and RNA are the polynucleotides known to carry genetic information in life. Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important. This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo. Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1. Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones. Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA. All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA. Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo.

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol−1 of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Binding of RNA template to a complex of HIV-1 reverse transcriptase/primer/template

HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Radiation target analysis of RNA.

Ribozymes are polynucleotide molecules with intrinsic catalytic activity, capable of cleaving nucleic acid substrates. Large RNA molecules were synthesized containing a hammerhead ribozyme moiety of 52 nucleotides linked to an inactive leader sequence, for total lengths of either 262 or 1226 nucleotides. Frozen RNAs were irradiated with high energy electrons. Surviving ribozyme activity was determined using the ability of the irradiated ribozymes to cleave a labeled substrate. The amount of intact RNA remaining was determined from the same irradiated samples by scanning the RNA band following denaturing gel electrophoresis. Radiation target analyses of these data revealed a structural target size of 80 kDa and a ribozyme activity target size of 15 kDa for the smaller ribozyme, and 319 kDa and 16 kDa, respectively, for the larger ribozyme. The disparity in target size for activity versus structure indicates that, in contrast to proteins, there is no spread of radiation damage far from the primary site of ionization in RNA molecules. The smaller target size for activity indicates that only primary ionizations occurring in the specific active region are effective. This is similar to the case for oligosaccharides. We concluded that the presence of the ribose sugar in the polymer chain restricts radiation damage to a small region and prevents major energy transfer throughout the molecule. Radiation target analysis should be a useful technique for evaluating local RNA:RNA and RNA:protein interactions in vitro.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide.

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodon-independent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a species-specific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.