Eubacterial tmRNAs (10Sa RNAs) are unique because they function, at least in Escherichia coli, both as tRNA and mRNA (for a review, see Muto et al., 1998). These ∼360 ± 40-nt-long RNAs are charged with alanine at their 3′ ends by alanyl-tRNA synthetases or AlaRS (Komine et al., 1994; Ushida et al., 1994). Alanylation occurs thanks to the presence of the equivalent of the G3-U70 pair, the major identity element for the alanylation of canonical tRNAs (Hou & Schimmel, 1988; McClain & Foss, 1988). Bacterial tmRNAs also have a short reading frame coding for 9 to 27 amino acids, depending on the species. E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans-translation process (Tu et al., 1995; Keiler et al., 1996; Himeno et al., 1997; Withey & Friedman, 1999). In E. coli, this amino acid tag is cotranslationally added to polypeptides synthesized from mRNAs lacking a termination codon, and the added 11-amino-acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al., 1996).

The recent rapid advances that have been made both in cryo-electron microscopy (cryo-EM) and X-ray crystallography of bacterial ribosomes or their subunits (e.g., Stark et al., 1997a, 1997b; Ban et al., 1998; Malhotra et al., 1998) have led to a correspondingly rapid advance in our understanding of the three-dimensional (3D) arrangement in situ of the ribosomal RNA and protein molecules. In 1997 we published a model for the 16S rRNA (Mueller & Brimacombe, 1997a), which was fitted to a cryo-EM reconstruction at 20 Å resolution of the Escherichia coli 70S ribosome carrying tRNAs at the ribosomal A and P sites (Stark et al., 1997a). Subsequently, on the basis of the available RNA–protein interaction data (Mueller & Brimacombe, 1997b), we were able to fit the structure of ribosomal protein S7 as determined by X-ray crystallography (Hosaka et al., 1997; Wimberly et al., 1997) into our model in such a way as to satisfy both the biochemical data and the electron density of the EM reconstruction (Tanaka et al., 1998). More recently, the 16S model has been refined to fit an EM reconstruction at 13 Å resolution (Brimacombe et al., 2000), the latter being itself a refinement of the published EM reconstruction at 18 Å (Stark et al., 1997b) of 70S ribosomes carrying an EF-Tu/tRNA ternary complex stalled with the antibiotic kirromycin. In the refined 16S model, only minor changes needed to be made in the arrangement of the rRNA region interacting with S7 and in the positioning of the protein itself.

The trpRNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2–3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP–RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5′-(UAGCC)-3′ repeated 11 times and measured their effect on the TRAP–RNA interaction. Deoxyribonucleoside substitutions revealed that a 2′-hydroxyl group (2′-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP–RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2′-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability. Recent results suggest a model with three main points. First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events. Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains. Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p. If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation. Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA. This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly.

In most species the 3′ splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3′ splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3′ splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.

In two recent articles by Nitta et al. (1998a, 1998b), evidence was presented that in vitro-transcribed protein-free 23S rRNA of Escherichia coli (and its individual domains) can catalyze peptide bond formation. The implications of these studies are considerable because demonstration of catalytic activity associated with protein-free 23S rRNA would be fundamental to our understanding of the mechanism and evolution of protein synthesis (Noller et al., 1992; Khaitovich et al., 1999).

“Crystallization, as applied to proteins, was long held in high esteem, but hardly considered science.” I quote from the preface to Alexander McPherson's new survey of the field, Crystallization of Biological Macromolecules. Years ago, McPherson's previous monograph (Preparation and Analysis of Protein Crystals, 1982, Wiley) introduced me to the subject. The growth of knowledge about protein crystallization in the past 17 years is vividly illustrated by comparison of the two books. The slim old volume contained a single chapter on experimental procedures for protein crystallization. In contrast, the 1999 book has three chapters on experimental issues, plus several chapters that review the emerging understanding of the physical chemistry of macromolecular crystallization.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron–rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.

Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916–923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926–1936). However, we observed that a U1 5′ splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915–926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U11 5′ splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.

Divalent metal ions are essential for the folding and catalytic activities of many RNAs. A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of RP α-phosphorothioate (PS) interference by the addition of soft divalent metal ions. To access the ability of such experiments to accurately identify metal-ion coordinations within a complex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4–P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Cate et al., Nat Struct Biol, 1997, 4:553]. We used a native gel mobility-shift to assay for P4–P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (≥0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4–P6 crystal structure. To control for such effects, 2′-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM). This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals. Both sites that were not specifically rescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though again phosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4–P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.

Life today requires the transmission of genetic information in the form of DNA, and a large number of catalytic and structural functions carried out by proteins. Without DNA to specify useful sequences of amino acids, functional proteins would not exist. Without proteins to carry out high-fidelity DNA replication and gene expression, DNA could not serve as the genetic material. How could either arise without the other?

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5′ end. Here, experimental evidence is presented for the existence of metastable structures in the 5′ leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5′ ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

Small nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3′ cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G[bull ]U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs that are translationally dormant or masked until meiotic maturation. Activation of the oocyte by fertilization leads to translational activation of the abundant cyclin and ribonucleotide reductase mRNAs at a time when they undergo cytoplasmic polyadenylation. In vitro unmasking assays have defined U-rich regions located approximately centrally in the 3′ UTRs of these mRNAs as translational masking elements. A clam oocyte protein of 82 kDa, p82, which selectively binds the masking elements, has been proposed to act as a translational repressor. Importantly, mRNA-specific unmasking in vitro occurs in the absence of poly(A) extension. Here we show that clam p82 is related to Xenopus CPEB, an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPEs) of maternal mRNAs and promotes their polyadenylation. Cloned clam p82/CPEB shows extensive homology to Xenopus CPEB and related polypeptides from mouse, goldfish, Drosophila and Caenorhabditis elegans, particularly in their RNA-binding C-terminal halves. Two short N-terminal islands of sequence, of unknown function, are common to vertebrate CPEBs and clam p82. p82 undergoes rapid phosphorylation either directly or indirectly by cdc2 kinase after fertilization in meiotically maturing clam oocytes, prior to its degradation during the first cell cleavage. Phosphorylation precedes and, according to inhibitor studies, may be required for translational activation of maternal mRNA. These data suggest that clam p82 may be a functional homolog of Xenopus CPEB.

Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3′ untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA.

Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5′-untranslated region, generally known as the IRES (for “internal ribosome entry site”). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES.

We have shown previously that directed hydroxyl radical probing of 16S rRNA from Fe(II) tethered to specific sites within the RNA gives valuable information about RNA–RNA proximities in 70S ribosomes. Here, we extend that study and present probing data from nt 424 in 16S rRNA. To tether an Fe(II) to position 424 in the rRNA we created a specific discontinuity in the RNA by in vitro transcription of the RNA as two separate fragments corresponding to nt 1–423 and 424–1542. An Fe(II)-BABE was covalently attached to a 5′-guanosine-α-phosphorothioate at position 424 and 30S subunits were reconstituted from the two pieces of rRNA and the small subunit proteins. Reconstituted 30S subunits capable of associating with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved positions in the RNA backbone that were close in three-dimensional space to the Fe(II), and the sites of cleavage were identified using primer extension. Fe(II) tethered to position 424 induces cleavage around nt 424, 513, and 531 in the 5′-domain of 16S rRNA and around nt 1008, 1029, 1044, and 1208 in the 3′-domain of 16S ribosomal RNA. These data constrain the positions of the 420, 1015, 1030 and 1000/1040 helices, for which there is little structural information. Since the 5′- and 3′-domains of 16S rRNA constitute the body and head, respectively, of 30S subunits, these findings provide direct evidence for proximity of RNA elements in the body and head of 30S.

A set of free energy values is suggested for RNA H-pseudoknot loops. The parameters are adjusted to be consistent with the theory of polymer thermodynamics and known data on pseudoknots. The values can be used for estimates of pseudoknot stabilities and computer predictions of RNA structures.