Probing RNA structures with hydroxyl radicals.

Fe(II)-EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar-phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure.

Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L.

Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages.

The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria. These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage. The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands.

Protein components contribute to active site architecture for eukaryotic ribonuclease P.

In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes.

Functional recognition of fragmented operator sites by R17/MS2 coat protein, a translational repressor.

The R17/MS2 coat protein serves as a translational repressor of replicase by binding to a 19 nt RNA hairpin containing the Shine-Dalgarno sequence and the initiation codon of the replicase gene. We have explored the structural features of the RNA operator site that are necessary for efficient translational repression by the R17/MS2 coat protein in vivo . The R17/MS2 coat protein efficiently directs lysogen formation for P22 R17 , a bacteriophage P22 derivative that carries the R17/MS2 RNA operator site within the P22 phage ant mRNA.

Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation.

Phosphorylation is important in the regulation of many cellular processes, yet the precise role of protein phosphorylation for many RNA-binding protein substrates remains obscure. In this report, we demonstrate that phosphorylation of a recombinant human immunodeficiency virus type-1 Rev protein promotes rapid formation of an efficient RNA-binding state. The apparent dissociation constant for ligand binding is enhanced 7-fold for the protein following phosphorylation; however, phosphate addition leads to a 1.

Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded RNA-binding activities.

RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops lytically following infection of these strains.

Efficient modification of RNA by porphyrin cation photochemistry: monitoring the folding of coaxially stacked RNA helices in tRNA(Phe) and the human immunodeficiency virus type 1 rev response element RNA.

Coaxially stacked RNA helices are a determined of RNA tertiary structure, but their presence is rarely detected using conventional chemical modification methods. In this report we describe a porphyrin ion photoreaction that enables one to monitor RNA stacking interactions and the folding of coaxially stacked RNA helices. The porphyrin cations meso-tetrakis(4-N-methylpyridyl)porphine, meso-tetrakis-(para-N-trimethylanilinium)porphine, and meso-tetrakis(2-N-methylpyridyl)porphine were used to characterize tRNA(Phe) and the human immunodeficiency virus type-I Rev response element RNA.

Ribonuclease P of Tetrahymena thermophila.

Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA. The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro. The role of the subunits in eucaryotes has not yet been established.

Improved method for selecting RNA-binding activities in vivo.

RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo.

Direct genetic selection for a specific RNA-protein interaction.

The decision between lytic and lysogenic development of temperate DNA bacteriophages is determined largely by transcriptional regulation through DNA-binding proteins. To determine whether a heterologous RNA-binding activity could control the developmental fate of a DNA bacteriophage, a derivative of P22 was constructed in which the chosen developmental pathway is regulated by an RNA-binding molecule interacting with its RNA target site located in a phage mRNA. In the example presented, lysogenic development of the phage relies upon R17 coat protein expression in the susceptible host cell and the availability of a suitable coat protein binding site encoded by the phage genome.

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al.

Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.

Fe(II)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higher-order folding of the group IB intron from Tetrahymena thermophila pre-rRNA. This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass. Significant portions of the phylogenetically conserved secondary structure are protected from Fe(II)-EDTA cleavage.

Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex.

Telomeres of Oxytricha nova macronuclear chromosomes consist of a repeated T4G4 sequence, single-stranded at the 3' terminus, bound by a heterodimeric protein. The cloning of genes for the two polypeptides and their separate expression in E. coli have enabled evaluation of their individual contributions to DNA binding.

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed.

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms.

Fe(II)-EDTA catalyzes the cleavage of nucleic acids with little or no base-sequence specificity. We have now studied the preference of this reagent in catalyzing the cleavage of single- versus double-stranded nucleic acid structures. Three RNA and two DNA molecules, each expected to contain both single- and double-stranded regions, were synthesized and their structures characterized by enzymatic digestion using secondary structure specific nucleases.

Two versions of the gene encoding the 41-kilodalton subunit of the telomere binding protein of Oxytricha nova.

Macronuclear chromosomes of the ciliated protozoan Oxytricha nova terminate with a single-stranded (T4G4)2 overhang. The (T4G4)2 telomeric overhang is tenaciously bound by a protein heterodimer. We have cloned and sequenced the gene encoding the 41-kDa subunit of this telomere binding protein.

Regulatory elements within the murine leukemia virus enhancer regions mediate glucocorticoid responsiveness.

Enhancer elements within nonleukemogenic (Akv) and T-cell leukemogenic (SL3-3) murine leukemia viruses demonstrate strong cell type preference in transcriptional activity. These transcription elements are additionally regulated by the synthetic glucocorticoid dexamethasone, and this pattern of regulation varies according to cell type. The sequences required for dexamethasone regulation for both Akv and SL3-3 are shown to include a 17-nucleotide consensus sequence previously termed the glucocorticoid response element (GRE).

Glucocorticoid regulation of murine leukemia virus transcription elements is specified by determinants within the viral enhancer region.

The transcriptional control region (the long terminal repeat, LTR) of the leukemogenic murine retrovirus SL3-3 contains a glucocorticoid-responsive consensus sequence, as does the corresponding region of the nonleukemogenic virus Akv. Dexamethasone increases gene expression directed by both LTR sequences. However, the responses of the LTRs of the two viruses to dexamethasone differ according to the cell line in which the response is measured.

Tissue-specific transcription preference as a determinant of cell tropism and leukaemogenic potential of murine retroviruses.

Inoculation of susceptible strains of mice with the SL3-3 strain of murine leukaemia viruses induces T-cell lymphomas, whereas injection of the Akv strain does not. Recombinant viruses that contain the long terminal repeat (LTR) of the SL3-3 virus and the gag, pol and env genes of the Akv virus are also leukaemogenic. The cell-type specificity of leukaemias induced by viruses containing different LTR sequences is due in part to the ability of the virus to replicate in the appropriate cellular environment.

Determination of the leukaemogenicity of a murine retrovirus by sequences within the long terminal repeat.

Although the murine retrovirus SL3-3 is highly leukaemogenic, in both the structure of its genome and in its properties of replication in tissue culture it closely resembles the nonleukaemogenic retrovirus Akv (refs 3, 4). An earlier investigation of the properties of recombinant SL3-3-Akv viruses localized the major determinant of leukaemogenicity outside the env gene, in a region of the viral genome that includes the gag gene and the noncoding long terminal repeat (LTR). To localize the determinant of SL3-3's leukaemogenicity more precisely we have now construced a recombinant provirus containing the LTR of SL3-3 and the coding region of Akv.