RNomics: identification and function of small non-protein-coding RNAs in model organisms.

In the recent past, our knowledge on small non-protein-coding RNAs (ncRNAs) has exponentially grown. Different approaches to identify novel ncRNAs that include computational and experimental RNomics have led to a plethora of novel ncRNAs. A picture emerges, in which ncRNAs have a variety of roles during regulation of gene expression. Thereby, many of these ncRNAs appear to function in guiding specific protein complexes to target nucleic acids. The concept of RNA guiding seems to be a widespread and very effective regulatory mechanism. In addition to guide RNAs, numerous RNAs were identified by RNomics screens, lacking known sequence and structure motifs; hence no function could be assigned to them as yet. Future challenges in the field of RNomics will include elucidation of their biological roles in the cell.

The IC-SNURF-SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A.

The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the coordinated control of an imprinting center (IC) at the 5' end of the SNURF-SNRPN gene. One gene cluster spans the centromeric part of this domain and contains several genes that are transcribed from the paternal chromosome only (MKRN3, MAGEL2, NDN, SNURF-SNRPN, HBII-13, HBII-85 and HBII-52). Apart from the HBII small nucleolar RNA (snoRNA) genes, each of these genes is associated with a 5' differentially methylated region (DMR). The second gene cluster maps to the telomeric part of the imprinted domain and contains two genes (UBE3A and ATP10C), which in some tissues are preferentially expressed from the maternal chromosome. So far, no DMR has been identified at these loci. Instead, maternal-only expression of UBE3A may be regulated indirectly through a paternally expressed antisense transcript. We report here that a processed antisense transcript of UBE3A starts at the IC. The SNURF-SNRPN sense/UBE3A antisense transcription unit spans more than 460 kb and contains at least 148 exons, including the previously identified IPW exons. It serves as the host for the previously identified HBII-13, HBII-85 and HBII-52 snoRNAs as well as for four additional snoRNAs (HBII-436, HBII-437, HBII-438A and HBII-438B), newly identified in this study. Almost all of those snoRNAs are encoded within introns of this large transcript. Northern blot analysis indicates that most if not all of these snoRNAs are indeed expressed by processing from these introns. As we have not obtained any evidence for other genes in this region, which, from the mouse data appears to be critical for the neonatal Prader-Willi syndrome phenotype, a lack of these snoRNAs may be causally involved in this disease.

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse.

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:

A novel brain-specific box C/D small nucleolar RNA processed from tandemly repeated introns of a noncoding RNA gene in rats.

Antisense box C/D small nucleolar RNAs (snoRNAs) guide the 2'-O-ribose methylations of eukaryotic rRNAs and small nuclear RNAs (snRNAs) through formation of a specific base pairing at each RNA methylation site. By analysis of a box C/D snoRNA cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to rRNA or an snRNA and exhibits a brain-specific expression pattern. It is uniformly expressed in all major areas of adult rat brain (except for choroid plexus) and throughout rat brain ontogeny but exclusively detected in neurons in which it exhibits a nucleolar localization. In vertebrates, known methylation guide snoRNAs are intron-encoded and processed from transcripts of housekeeping genes. In contrast, RBII-36 snoRNA is intron-encoded in a gene preferentially expressed in the rat central nervous system and not in proliferating cells. Remarkably, this host gene, which encodes a previously reported noncoding RNA, Bsr, spans tandemly repeated 0.9-kilobase units including the snoRNA-containing intron. The novel brain-specific snoRNA appears to result not only from processing of the debranched lariat but also from endonucleolytic cleavages of unspliced Bsr RNA (i.e. an alternative splicing-independent pathway unreported so far for mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA were exclusively detected in the Rattus genus of rodents, suggesting a very recent origin of this brain-specific snoRNA.

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel.

Propafenone has been shown to affect the delayed-rectifier potassium currents in cardiomyocytes of different animal models. In this study we investigated effects and mechanisms of action of propafenone on HERG potassium channels in oocytes of Xenopus laevis with the two-electrode voltage-clamp technique. Propafenone decreased the currents during voltage steps and the tail currents. The block was voltage-dependent and increased with positive going potentials (from 18% block of tail current amplitude at -40 mV to 69% at +40 mV with 100 micromol/l propafenone). The voltage dependence of block could be fitted with the sum of a monoexponential and a linear function. The fractional electrical distance was estimated to be delta=0.20. The block of current during the voltage step increased with time starting from a level of 83% of the control current. Propafenone accelerated the increase of current during the voltage step as well as the decay of tail currents (time constants of monoexponential fits decreased by 65% for the currents during the voltage step and by 37% for the tail currents with 100 micromol/l propafenone). The threshold concentration of propafenone effect was around 1 micromol/l and the concentration of half-maximal block (IC50) ranged between 13 micromol/l and 15 micromol/l for both current components. With high extracellular potassium concentrations, the IC50 value rose to 80 degrees mol/l. Acidification of the extracellular solution to pH 6.0 increased the IC50 value to 123 micromol/l, alkalization to pH 8.0 reduced it to 10 micromol/l and coexpression of the beta-subunit minK had no statistically significant effect on the concentration dependence. In conclusion, propafenone has been found to block HERG potassium channels. The data suggest that propafenone affects the channels in the open state and give some hints for an intracellular site of action.

A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15.

Balanced translocations affecting the paternal copy of 15q11--q13 are a rare cause of Prader-Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3' exons of this gene. Using RT--PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype.

Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization.

We have identified three C/D-box small nucleolar RNAs (snoRNAs) and one H/ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded in the brain-specific serotonin 2C receptor gene. The three human C/D box snoRNAs map to chromosome 15q11-q13, within a region implicated in the Prader-Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2'-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA.

In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.

Selenocysteine inserting RNA elements modulate GTP hydrolysis of elongation factor SelB.

Elongation factor SelB is required for the incorporation of the amino acid selenocysteine into proteins in Escherichia coli. Selenocysteine incorporation is thought to be achieved by simultaneous binding of SelB to selenocysteyl-tRNASec and to an mRNA hairpin structure located 3' adjacent to the UGA selenocysteine codon. SelB was shown previously to bind to GTP or GDP in a molar ratio of 1:1. Here, we demonstrate that SelB, like EF-Tu, exhibits a low intrinsic GTPase activity in the absence of ribosomes. As shown for EF-Tu, GTPase activity of SelB is stimulated by the presence of E. coli 70S ribosomes; the apparent K(m) for GTP hydrolysis is 55 microM. Interestingly, in the presence of the mRNA hairpin which promotes selenocysteine incorporation, GTPase activity of SelB increases additionally by 3-4-fold; stimulation is due to kcat increasing from 0.05/min in the absence to 0.16/min in the presence of the mRNA hairpin. This mRNA-induced stimulation of SelB GTPase activity depends on the presence of ribosomes. The minimal region of the mRNA hairpin capable to stimulate GTP hydrolysis by SelB locates within the upper half of the hairpin; this part of the mRNA structure was demonstrated previously to be sufficient for binding of the mRNA to SelB. On the basis of these results, we propose that binding of the mRNA hairpin to SelB induces a conformational switch within SelB thereby promoting an increase in ribosome-mediated GTP hydrolysis.

In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB.

The special elongation factor SelB of Escherichia coli promotes selenocysteine incorporation into formate dehydrogenases. This is thought to be achieved through simultaneous binding to selenocysteyl-tRNASec and, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin structure 3' adjacent to the UGA selenocysteine codon. By in vitro selection, novel RNA sequences ("aptamers"), which can interact tightly and specifically with SelB, were isolated from an RNA library. The library was comprised of mutagenized variants of the wild-type fdhF mRNA hairpin. One-half of the selected sequences contained the apical stem-loop of the fdhF mRNA hairpin highly conserved. Some of the aptamers showed deviations in the primary sequence within this region of the wild-type fdhF hairpin motif while still binding with high affinity to SelB. Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been selected. To dissect SelB binding to the fdhF hairpin from the overall biological function of this complex, four selected aptamers were analyzed in vivo for UGA readthrough in a lacZ fusion construct. Among these, one promoted UGA readthrough in vivo. Three of the aptamers, however, were drastically reduced or unable to replace the fdhF mRNA hairpin in vivo, despite the similar secondary structure and binding affinities of these RNAs compared with the wild-type motif. This finding implies functions of the fdhF hairpin that go beyond the mere tethering of selenocysteyl-tRNASec to the UGA codon.

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.

In vitro selection analysis of neomycin binding RNAs with a mutagenized pool of variants of the 16S rRNA decoding region.

An in vitro selection for neomycin B binding was carried out with an RNA pool containing a 47-nucleotide domain of the decoding region of 16S ribosomal RNA, mutated at 30% per base position. The degenerate region was comprised of an oligonucleotide analogue ("motif A") of the decoding region in 30S subunits which has previously been shown to interact with the aminoglycoside antibiotic neomycin B and tRNA ligands. After five cycles of selection/amplification, RNA sequences were isolated which specifically bound to neomycin B. Cloning and sequencing showed that none of the isolated clones shared primary sequence or secondary structure homology with the decoding region of 16S RNA. Instead, a new set of sequences was isolated which could be folded into a defined hairpin structure designated as motif B. We investigated the affinity of motif A, motif B, the unselected pool RNA, and the corresponding unmutagenized "parent" RNA to neomycin B at different Mg2+ concentrations. Under buffer conditions of low ionic strength all RNAs tested bound nonspecifically to neomycin B. However, motif B bound to neomycin B at Mg2+ concentrations at which binding of the other RNAs tested was significantly lower or not detectable. This is consistent with motif B exhibiting a higher affinity for neomycin B than motif A under these conditions. Motif B has previously been isolated from an in vitro selection to identify RNA sequences with affinity to neomycin B using a completely randomized RNA pool which shared no relationship to motif A. Our results indicate that motif B might represent a highly optimized RNA sequence for neomycin B binding; conversely, the A-site motif in 16S rRNA (motif A) might not be an optimal target for neomycin B recognition.

Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB.

On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.

Structure and function of ribosomal RNA.

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.

Cleavage of tRNA by Fe(II)-bleomycin.

We have investigated the action of the chemotherapeutic agent Fe(II)-bleomycin on yeast tRNA(Phe), an RNA of known three-dimensional structure. In the absence of Mg2+ ions, the RNA is cleaved preferentially at two major positions, A31 and G53, both of which are located at the terminal base pairs of hairpin loops, and coincide with the location of tight Mg2+ binding sites. A fragment of the tRNA (residues 47-76) containing the T stem-loop is also cleaved specifically at G53. Cleavage of both the intact tRNA and the tRNA fragment is abolished in the presence of physiological concentrations of Mg2+ (> 0.5 mM). Since Fe(II) is not displaced from bleomycin under these conditions, we infer that tight binding of Mg2+ to tRNA excludes productive interactions between Fe(II)-bleomycin and the RNA. These results also show that loss of cleavage is not due to Mg(2+)-dependent formation of tertiary interactions between the D and T loops. In contrast, cleavage of synthetic DNA analogs of the anticodon and T stem-loops is not detectably inhibited by Mg2+, even at concentrations as high as 50 mM. In addition, the site specificities observed in cleavage of RNA and DNA differ significantly. From these results, and from similar findings with other representative RNA molecules, we suggest that the cleavage of RNA by Fe(II)-bleomycin is unlikely to be important for its therapeutic action.

Hydroxyl radical cleavage of tRNA in the ribosomal P site.

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.