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Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer. A 22-nucleotide fragment of a transfer RNA regulates translation by binding to the mRNA of a ribosomal protein and increasing its expression, and downregulation of the fragment in patient-derived liver tumour cells reduces tumour growth in mice. The functional roles of small RNA fragments derived from tRNAs are not well known, but evidence is growing that some play a part in various cellular processes. Mark Kay and colleagues show that a 22-nucleotide fragment from the 3′ end of leucine tRNA can regulate translation. The fragment binds to the mRNA of a ribosomal protein to upregulate its expression. When this interaction is suppressed in human cells in culture, cell death occurs. Decreasing the levels of the tRNA fragment with an antisense oligonucleotide can slow the growth of liver tumours in mice. Technologies aimed at reducing expression of this tRNA fragment might have utility in treating cancer.

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4–RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein–RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA–protein complexes. Three-colour fluorescence resonance energy transfer and molecular dynamics simulations are used to study the events occurring early in assembly of the 30S ribosome; within a non-native intermediate S4 ribosomal protein–16S RNA structure, S4 is capable of altering the RNA helix dynamics to facilitate conformation changes that enable subsequent protein binding. The assembly of protein–RNA complexes can involve the sculpting of RNA structure as proteins are sequentially bound, but direct demonstration of the details of these changes has been difficult. Sarah Woodson and colleagues have used sophisticated three-colour fluorescence resonance energy transfer (FRET) and modelling to look at the events occurring early in the assembly of the 30S ribosome. They find that when the S4 ribosomal protein interacts with the 16S rRNA, there is a stable, on-path, non-native intermediate, and that the S4 protein can alter the RNA helix dynamics to facilitate conformational changes that enable subsequent protein binding.

Ribosomes are translational machineries that catalyse protein synthesis. Ribosome structures from various species are known at the atomic level, but obtaining the structure of the human ribosome has remained a challenge; efforts to address this would be highly relevant with regard to human diseases. Here we report the near-atomic structure of the human ribosome derived from high-resolution single-particle cryo-electron microscopy and atomic model building. The structure has an average resolution of 3.6 A, reaching 2.9 A resolution in the most stable regions. It provides unprecedented insights into ribosomal RNA entities and amino acid side chains, notably of the transfer RNA binding sites and specific molecular interactions with the exit site tRNA. It reveals atomic details of the subunit interface, which is seen to remodel strongly upon rotational movements of the ribosomal subunits. Furthermore, the structure paves the way for analysing antibiotic side effects and diseases associated with deregulated protein synthesis. The structure of the human ribosome at high resolution has been solved; by combining single-particle cryo-EM and atomic model building, local resolution of 2.9 A was achieved within the most stable areas of the structure. This paper presents the near-atomic structure of the human ribosome determined using single-particle cryo-electron microscopy and atomic model building. The structure reaches the high resolution of 2.9 A in the most stable regions of the complex, allowing the visualization of previously inaccessible elements, such as regions of the ribosomal RNA scaffolding and amino acid side chains. In addition, the significant remodelling of the interface between the large and small subunits is clarified.

Emerging evidence suggests that the ribosome has a regulatory function in directing how the genome is translated in time and space. However, how this regulation is encoded in the messenger RNA sequence remains largely unknown. Here we uncover unique RNA regulons embedded in homeobox (Hox) 5' untranslated regions (UTRs) that confer ribosome-mediated control of gene expression. These structured RNA elements, resembling viral internal ribosome entry sites (IRESs), are found in subsets of Hox mRNAs. They facilitate ribosome recruitment and require the ribosomal protein RPL38 for their activity. Despite numerous layers of Hox gene regulation, these IRES elements are essential for converting Hox transcripts into proteins to pattern the mammalian body plan. This specialized mode of IRES-dependent translation is enabled by an additional regulatory element that we term the translation inhibitory element (TIE), which blocks cap-dependent translation of transcripts. Together, these data uncover a new paradigm for ribosome-mediated control of gene expression and organismal development.

Ribosomes, the cell's protein-synthesis machines, are assembled from their components in a defined order. It emerges that the first assembly step must overcome dynamic structural rearrangements. See Article p.334 The assembly of protein–RNA complexes can involve the sculpting of RNA structure as proteins are sequentially bound, but direct demonstration of the details of these changes has been difficult. Sarah Woodson and colleagues have used sophisticated three-colour fluorescence resonance energy transfer (FRET) and modelling to look at the events occurring early in the assembly of the 30S ribosome. They find that when the S4 ribosomal protein interacts with the 16S rRNA, there is a stable, on-path, non-native intermediate, and that the S4 protein can alter the RNA helix dynamics to facilitate conformational changes that enable subsequent protein binding.

Three studies have characterized the full complement of RNA folding in cells. They find large numbers of secondary structures in RNA, some of which may have functional consequences for the cell. See Letters p.696 , p.701 & p.706 Being single-stranded, RNA can adopt a diversity of secondary structures via inter- and intramolecular base-pairing. Three studies published in this issue of Nature provide an in-depth view of the variety, dynamics and functional influence of RNA structures in vivo. Sarah Assmann and colleagues map the in vivo RNA structure of over 10,000 transcripts in the model plant Arabidopsis thaliana. Their struc-seq (structure-seqence) approach incorporates in vivo chemical (DMS) probing and next-generation sequencing to provide single-nucleotide resolution on a genome-wide scale. Distinct patterns of structure are found to be correlated with coding regions, splice sites and polyadenylation sites. Comparison of these results with those obtained by earlier technologies reveals that, although predictions for some classes of genes were fairly accurate, others, such as those involved in stress response, were poorly predicted and may reflect changes that made them more adapted to that condition. Jonathan Weissman and colleagues have also developed a DMS-seq method to globally monitor RNA structure to single-nucleotide precision in yeast and mammalian cells. Comparing their findings with in vitro data, the authors conclude that there is less structure within cells than expected. Even thermostable RNA structures can be denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Howard Chang and colleagues asked a different question: how does RNA secondary structure change on a transcriptome-wide level in related individuals? By calculating the RNA secondary structures of two parents and their child, they find that about 15% of transcribed single-nucleotide variants affect local secondary structure. These 'RiboSNitches' are depleted in certain locations, suggesting that a particular RNA structure at that site is important. This study illustrates that there is much to be learned about how changes in RNA structure, particularly as imparted by genetic variation, can alter gene expression.

The emergence of the ribosome constituted a pivotal step in the evolution of life. This event happened nearly four billion years ago, and any traces of early stages of ribosome evolution are generally thought to have completely eroded away. Surprisingly, a detailed analysis of the structure of the modern ribosome reveals a concerted and modular scheme of its early evolution.

Group I and II introns are mobile elements that propagate by insertion into different genes. Some introns of both types self-splice in vitro by transesterification reactions catalysed by the intron RNA. These transesterifications are reversible, and it has been suggested that reverse splicing followed by reverse transcription and recombination with genomic DNA may be a mechanism for intron transposition. In vivo the splicing of many, if not all, group I and II introns requires protein factors, which may facilitate correct folding of the intron RNAs. Here we show that the Neurospora mitochondrial large rRNA intron, a group I intron that is not self-splicing in vitro, undergoes reverse splicing in a reaction promoted by the CYT-18 protein, the Neurospora mitochondrial tyrosyl-tRNA synthetase, which is required for splicing the intron in vivo. In contrast to known RNA-catalysed reverse splicing reactions, this protein-assisted reverse splicing is sufficiently rapid to compete with forward splicing at low RNA concentrations under physiologically relevant conditions, including high GTP and low Mg2+ concentrations. Our results indicate that proteins that promote splicing could contribute to intron mobility by promoting reverse splicing in vivo.

The ribosome is a particle made of RNA and protein that is found in abundance in all cells that are actively making protein. It catalyses the messenger RNA-directed synthesis of proteins. Recent structural work has demonstrated a profound involvement of the ribosome's RNA component in all aspects of its function, supporting the hypothesis that proteins were added to the ribosome late in its evolution.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein–RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

The 'RNA world' hypothesis proposes an early stage in the evolution of life in which both genomic and catalytic functions were fulfilled by RNA. The evolution of RNA-catalysed protein synthesis would have been a necessary step in the transition from such an RNA world to modern protein-dominated biology. For this to have been possible, RNA must be capable of catalysing amide-bond formation using acylated carrier RNA substrates as amino-acid donors. We have used in vitro selection and evolution to isolate ribozymes with acyl transferase activity from a pool of random RNA sequences. One of these acyl transferases with a 5'-amino group transfers an amino acid to itself in a reaction that we propose to be analogous to peptidyl transfer on the ribosome.

UNDERSTANDING the process whereby the ribosome translates the genetic code into protein molecules will ultimately require high-resolution structural information, and we report here the first crystal structure of a protein from the small ribosomal subunit. This protein, S5, has a molecular mass of 17,500 and is highly conserved in all lifeforms1–4. The molecule contains two distinct α/β domains that have structural similarities to several other proteins that are components of ribonucleoprotein complexes. Mutations in S5 result in several phenotypes which suggest that S5 may have a role in translational fidelity and translocation. These include ribosome ambiguity or ram5, reversion from streptomycin dependence6 and resistance to spectinomycin6. Also, a cold-sensitive, spectinomycin-resistant mutant of S5 has been identified which is defective in initiation7. Here we show that these mutations map to two distinct regions of the molecule which seem to be sites of interaction with ribosomal RNA. A structure/function analysis of the molecule reveals discrepancies with current models8,9 of the 308 subunit.

Direct chemical 'footprinting' shows that translocation of transfer RNA occurs in two discrete steps. During the first step, which occurs spontaneously after the formation of the peptide bond, the acceptor end of tRNA moves relative to the large ribosomal subunit resulting in 'hybrid states' of binding. During the second step, which is promoted by elongation factor EF-G, the anticodon end of tRNA, along with the messenger RNA, moves relative to the small ribosomal subunit.

Sequence information for four RNA ribosome binding sites has been obtained from phiX174 phiRNA synthesised in vitro. The ribosome binding sites have been assigned to sites on the phiX174 genome by comparison with known DNA sequence data.

One of the most enigmatic features of eukaryotic ribosomal RNA is the presence of many methylated nucleotides. The numbers of RNA methyl groups range from approximately 70 per ribosome in yeast1 to over 100 in vertebrates2,3. Here it is shown that the methylated nucleotides in Xenopus laevis rRNA are broadly but non-uniformly distributed. In 18S rRNA 2′-O-methylations are partly concentrated in the 5′ region and base methylations near the 3′ end. In 28S rRNA methyl groups are infrequent in the 5′ region, moderately frequent in the central region and abundant in an 1,100-nucleotide tract near the 3′ end.

SINCE the original observation of Perry1 that a low concentration of actinomycin D (0.04 μg ml−1) selectively inhibits ribosomal RNA (rRNA) synthesis in vivo, this concentration of drug has been used by numerous investigators to selectively inhibit the synthesis of rRNA in cells in culture. Since nucleolar ribosomal DNA contains a high amount of deoxyguanosine and deoxycytosine bases2 and actinomycin D selectively binds to deoxyguanosine3, it has been thought that actinomycin D preferentially binds to nucleolar DNA to inhibit the transcription of 45S ribosomal RNA precursor.

The nucleotide sequence spanning the ribosomal RNA (rRNA) genes of cloned human mitochondrial DNA reveals an extremely compact genome organization wherein the putative tRNA genes are probably ‘butt-jointed’ around the two rRNA genes. The sequences of the rRNA genes are significantly homologous in some regions to eukaryotic and prokaryotic sequences, but distinctive ; the tRNA genes also have unusual nucleotide sequences. It seems that human mitochondria did not originate from recognizable relatives of present day organisms.

Purified RNA polymerase II from calf thymus can bind to about 15% of the nucleosome core particles prepared from mouse myeloma cells, forming a discrete complex having a sedimentation coefficient of 18S. These bound nucleosome cores are heavily enriched in transcribed DNA sequences, are deficient in histones H2A and H2B, and undergo a reversible change in structure on RNA polymerase II binding.

THERE is no doubt that the large ribosomal RNAs play specific roles in ribosome function1. Yet the thrust of experimentation during the past 5 years indicates clearly that the biologist tends to view these roles as structural, function being reserved by and large for the ribosomal protein components2,3. Whether or not this prejudice can be maintained remains to be seen. In any case, the existence of an approximate sequence for a 16S rRNA provides a basis for detailed characterisations of this molecule3,4. Here we begin to define 16S rRNA function by localising the major conserved regions in the molecule through a comparative analysis of 27 prokaryotic 16S rRNA primary structures.

Experiments involving rifampicin show that 5S RNA is generated from a transcription unit carrying between thirteen and thirty times the amount of genetic information necessary for the production of one molecule.

The sequencing of the ribosome binding sites and parts of the coat protein cistron has enabled the order of the three R17 genes to be established by direct chemical means. Translation may be influenced by the conformation of the RNA.

Ribosome Formation from Subunits: Dependence on Formylmethionyl-transfer RNA in Extracts from E. coli

Functional 30S ribosomal subunits can be reconstructed from the 16S ribosomal RNA of one species of bacteria and the ribosomal proteins from a distantly related species.

A transcription factor, Ψ r , which preferentially stimulates the synthesis of ribosomal RNA in vitro has been identified in crude extracts of E. coli. This factor activity is also a component of Qβ replicase.

The ‘bobbed’ (bb) mutation in Drosophila is represented phenotypically by shortened and abnormally thin scutellar bristles and by delayed development. There is a direct correlation between bristle size and ribosomal RNA (rRNA) synthesis1, and the bb mutation was at first explained as a deficiency of rRNA genes (rDNA)2. However, the bb phenotype can occur in Drosophila melanogaster1 and Drosophila hydei3 with high rDNA content, while phenotypically wild-type flies are known with few rRNA genes, suggesting that what matters is not the number of rRNA genes but their transcriptional activity. In D. melanogaster, it has recently emerged that rRNA genes interrupted by an intervening sequence are not transcribed4. We now report that in D. hydei, the length of the scutellar bristle is directly proportional to the number of rRNA genes without this intervening sequence.

A METHOD has already been described for isolating RNA from the small subunit of the E. coli ribosome which had altered conformation by the following criteria: additional protein binding sites1,2, sensitivity to T1 RNase3 and mobility on polyacrylamide–Agarose electrophoresis3. We also showed that it was possible to detect the conformational difference by electron microscopy4. Even though the details of the structure were either below the resolution of the electron microscope, or not sufficiently homogeneous to describe in detail, length measurements of the molecules gave a statistically significant description of the structure. Previously, the only significant ribosomal RNA structure that could be resolved in the electron microscope was the characteristic secondary structure of RNA of the large subunit of eukaryotic ribosomes, or the precursor to eukaryote rRNA5. We wished to find out whether, generally, in rRNAs other than the 16S of E. coli, one could detect additional structure in the electron microscope, the details of which may be beyond the resolving power, and whether there are preparation-sensitive conformations in ribosomal RNA. The two preparation procedures used correspond to those described1, acetic acid-urea extraction (rRNAa) and the conventional phenol extraction (rRNAp). We show here that there are in fact previously unrecognised structural elements in a variety of ribosomal RNAs.

THE nucleoside analogue 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) inhibits the synthesis of heterogeneous nuclear RNA (hnRNA) at the initiation level in explanted salivary glands of Chironomus tentans, but does not interfere with the synthesis of ribosomal RNA and transfer RNA (refs 1–4). When the radioactive RNA precursors and the inhibitor are coincidently added to the glands the labelling of hnRNA transcripts produced by smaller transcriptional units is suppressed before that of the larger ones2,3. If, however, the glands are pretreated with DRB, and the time of preincubation is sufficiently long, labelling of small as well as large hnRNA transcripts is equally eliminated, and the DNA template becomes depleted of nascent hnRNA chains2–4. Important questions, arising from these findings, are whether DRB can be washed out from the cells, and whether a reinitiation of hnRNA synthesis can subsequently be achieved. This paper presents data which show that the initiation of the RNA synthesis resumes when glands pretreated with DRB are washed and replenished with inhibitor-free medium.

Since the initial discovery that the DNase I sensitivity of the globin genes in different cell types correlates with globin gene expression1, this relationship has been shown to hold true for a variety of genes, including the genes for ovalbumin2, conalbumin3, α- and β-globin in chicken4,5, several heat-shock proteins in Drosophila6,7, the r-chromatin of Tetrahymena8 and the viral polyoma minichromosome9. Although genes transcribed by RNA polymerases I and II have been studied extensively, the genes transcribed by RNA polymerase III have not. We have therefore investigated the DNase I sensitivity of transfer RNA (tRNA) and oogenetic 5S RNA genes in the liver and erythocyte nuclei of Xenopus laevis. The oogenetic 5S genes are not transcribed in any known somatic cell10,11, and tRNA genes are transcribed in the hepatocyte but are inactive in the erythrocyte. We show here that, although in these two cell types the correspondence between DNase I sensitivity and gene transcription holds good for globin and the ribosomal genes, the tRNA and oogenetic 5S genes are DNase I sensitive in both liver and erythrocyte nuclei. Thus for the genes transcribed by polymerase III the correspondence of sensitivity and expression breaks down.

WE have used in situ hybridisation to locate a major 5S RNA locus near the end of the long arm of chromosome 1 of man. This information represents a step towards the understanding of the synthesis, assembly and regulation of ribosomes. The 5S RNA molecule is associated with the larger ribosomal particle in both higher and lower organisms and is required for ribosome function. Ribosomal RNA—18S and 28S—is synthesised at the nucleolar organiser regions on the chromosomes of the D and G groups1. Genes coding for 5S RNA have been located in Drosophila2, Xenopus3 and several other organisms4, and our data now confirm preliminary reports5,6 that placed the 5S genes on chromosome 1 of man. We also suggest a mechanism for positioning the 5S genes near nucleoli at interphase.

Three large sections of 16S RNA, obtained by partial enzymatic hydrolysis, and together representing nearly one-fifth of the molecule, have been analysed. Knowledge of their primary structure allows features of the secondary structure to be envisaged.

An approach is described for determining secondary structure in large RNA molecules, in which families of short interacting RNA fragments are isolated and identified as such by two-dimensional gel electrophoresis. Data covering over half of the 16S ribosomal RNA from Escherichia coli are presented, including two long-range interactions.

THE concept of self-assembly has been shown to apply to the assembly of the Escherichia coli 30S ribosome in vitro by Nomura and his co-workers1,2. The reconstitution of functional 30S ribosomes from purified mature 16S RNA (m16 RNA in the nomenclature of Hecht and Woese3) and total 30S proteins has been studied in detail. The macromolecular information necessary for this in vitro assembly clearly resides in the component parts with no requirements for enzymes or cellular structures (such as membranes) which are not ultimately part of the 30S ribosome. The reconstitution system developed by Traub and Nomura1 has been useful in a variety of studies which have greatly aided our understanding of ribosome assembly, structure, and function. The biosynthesis of ribosomes, however, is known to differ in several ways from this in vitro reconstitution. The in vivo process is composed of a series of cooperative and sequential interactions between the primary ribosomal UNA transcripts and of ribosomal proteins, as well as the modification of both RNA and protein4,5.

Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes. These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA.

WE have sequenced the DNA region immediately preceding the structural gene for the 5S ribosomal RNA of yeast (Saccharomyces cerevisiae). Since the 5S RNA, a component of the larger ribosomal subunit, has a 5′ triphosphate1,2, this molecule is likely to be the direct product of the gene, unmodified by maturation at the 5′ end. Thus the DNA sequence immediately before this region must contain the recognition sequences where the RNA polymerase binds to begin synthesis, that is, this sequence must represent a eukaryotic promoter.

Messenger RNA was covalently linked to 18S ribosomal RNA in eukaryotic 40S and 80S initiation complexes by photoreaction with an RNA cross-linking agent, 4′-substituted psoralen. The sites of interaction of the mRNA capped 5′-region included some 3′-ends of 18S rRNA.

THE ribosomal RNA (rRNA) molecules both of prokaryotes and of eukaryotes are not the immediate products of DNA transcription. Rather, precursors of the mature RNA species undergo a series of nucleotide modifications and/or cleavage events1,2. Only limited information is available concerning the mechanics of and rationale for post-transcriptional tailoring of RNA because of the large sizes of the precursor molecules and consequent difficulty in evaluating their nucleotide sequences.

THE genes for the 23S and 16S rRNAs are physically linked1. Using the drugs rifampicin and actinomycin D, it has been shown that the 23S and 16S rRNA cistrons are co-transcribed and the promotor is proximal to 16S rRNA cistron2,3. Recent work with an Escherichia coli strain deficient in RNase III has provided further evidence for co-transcription, showing the presence of a long polycistronic RNA which could be cleaved to the immediate precursors of 23S and 16S rRNAs when treated with RNase III in vitro4. We have now studied the transcription of rRNA in E. coli using γ-32P-labelled triphosphates and have shown that the 16S rRNA precursor in our preparation has a 5′-pppA end and that in our system it is neither cleaved nor replaced by 5′-pA as observed by Takanami5. We have not found any detectable 5′-γ-32P-triphosphate end in the 23S rRNA species. This observation further verifies directly that both the 16S and 23S rRNAs belong to one transcriptional unit in the following order: promotor–16S–23S.

IN prokaryotes, the genes for 5S, 16S and 23S ribosomal RNA are linked in a polycistronic transcriptional unit1,2. In eukaryotes, on the other hand, the analogous unit contains the genes for 5.8S, 18S and 28S rRNAs, whereas the 5S RNA genes are found elsewhere in the genome. The analogous positions occupied by prokaryotic 5S RNA genes and eukaryotic 5.8S RNA in their respective genomes prompted Doolittle and Pace to speculate that these two RNA classes, both of which are components of the large ribosomal subunit, might have a common evolutionary origin3. If these RNAs are in fact homologous, one would expect their sequences to bear some relationship to each other. We report here our evaluation of possible structural relationships between known sequences of 5.8S and 5S rRNA, and conclude that they have no common evolutionary origin.

Methyl deficient 16S RNA from kasugamycin resistant Escherichia coli has been used to study methylation of RNA during ribosome assembly in vitro. Methylation occurs at an intermediate stage and is inhibited at a late stage of assembly. 30S ribosomal proteins required for methylation, and inhibition, have been identified.

Poliovirus RNA that had been labelled with 125I in the 5′-terminal protein (VPg) was found competent to form an initiation complex of translation in a cell-free reticulocyte lysate. In conditions of ribosome binding, no cleavage occurred between VPg and RNA. We conclude that removal of VPg from poliovirus RNA is not a prerequisite for this RNA to initiate translation in vitro.

IT has been proposed that a key event in the recognition of a prokaryotic protein initiation site by the ribosome is the interaction between the 3′-terminal sequence of the 16S rRNA and a purine-rich sequence preceding the initiator triplet by three to nine nucleotides1,2 This hypothesis predicts that if the 3′-terminal region of the 16S rRNA were blocked, binding of the ribosome to the initiator region of an mRNA should be diminished or prevented, while binding to the trinucleotide A-U-G should not be affected We have shown that the oligonucleotide A-G-A-G-G-A-G-G-UOH (P-2a), eight bases of which are complementary to the sequence A-C-C-U-C-C-U-U-AOH at the 3′-termmal region of 16S rRNA (ΔG = −16.9 kcal mol−1 calculated as described in Table 1), binds tightly to the 30S ribosome and is released as a complex with the 3′-terminal 49-mer of 16S rRNA after digestion with cloacin DF13 (ref. 3). We report here that this oligonucleotide either strongly inhibits Qβ RNA binding to 70S ribosomes or decreases the stability of the initiation complex formed in its presence As we found that the formation of complexes of pA-U-G and 70S ribosomes was, not inhibited by the oligonucleotide, we conclude that inhibition (or destabilisation) of Qβ RNA binding is not due to an effect on the interaction between fMet-tRNA and the initiation triplet or on the association of the 30S and 50S ribosome subunits.

The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. These elements can modulate gene expression, but it is not known whether they normally function in genetic control. To determine whether IS elements could exert control through specific RNA transcripts, we hybridised lambda NNC1857 r14 (carrying IS1) and pBR322 (carrying a portion of IS2) to Northern blots of E. coli RNA. Regions of homology between the IS elements and ribosomal RNA were observed. Computer analysis of reported nucleotide sequences detected large segments of homology between the IS elements and both 23S and 16S rRNA. Additional homologous sequences in phi X174 and a leader region of a ribosomal protein gene cluster were also detected. The homologous sequence between IS2 and 16S rTNA is the same sequence in phi X174 DNA which codes for the ends of the E and D gene and the start of J. The partial IS sequences may represent silent evolutionary remnants or they could modulate the expression of genes carrying these sequences.

THE conservative nature of bacterial ribosomal RNA (rRNA) is clear from the narrow range of its guanosine–cytosine (GC) content compared with the base composition of whole cell DNA. The majority of bacterial DNAs contain from 35 to 75 % GC, whereas the GC content of rRNA is restricted to a narrow range of 50–53% (ref. 1). Indeed, the ribosomal DNA (rDNA) of many bacterial species can be isolated from sheared preparations of whole cell DNA solely on the basis of its GC content. Furthermore, nucleic acid hybridisation has indicated considerable homology between the rRNAs of diverse bacterial species2–4 and helped provide phylogenetic support for taxo-nomic relationships. In an investigation of 22 different bacteria Pace and Campbell found that organisms whose rRNA showed closer homology to Escherichia coli rRNA, showed less rRNA homology to Bacillus stearothermophilus rRNA and vice versa2. Both sedimentation and gel electrophoresis studies in non-denaturing conditions have shown very small, if any, migratory differences between either the 23S or 16S rRNA species of different bacterial species5,6. By using a modified buffer system coupled with double labelling, we have found that both the large and small species of rRNA from Streptococcus faecalis, B. subtilis, and E. coli can be all distinguished on the basis of significant differences in their mobility in both composite polyacrylamide–Agarose gels and polyacrylamide gels.

In this article Dr Davis reviews recent developments in the search for the precise nature of the cycle of ribosomes in protein synthesis.

The conformations of RNA in yeast ribosomes and in the free state are similar. The ribosomal proteins are believed to be in the interior and associated with non-helical parts of the RNA and probably with each other; the surface is largely RNA.

The closed rRNA promoter and the protein synthesis elongation factor TuTs are prerequisites for the selective control of in vitro rRNA synthesis by ppGpp.

Phytohaemagglutinin stimulates the synthesis of the 45S ribosomal precursor in lymphocytes without concomitant protein synthesis; it also decreases the loss of newly synthesized 18S RNA that normally takes place in these cells at rest.

THE two large ribosomal RNA components (26S and 17S rRNA) of yeast exhibit a noteworthy structural similarity as judged by nucleotide composition1, the degree of methylation2, and the extensive cross hybridization with homologous DNA3. Because the 17S rRNA can compete with much more than 50 per cent of 26S rRNA in hybridization with homologous DNA3, and because the estimated chain length of the 26S rRNA is about twice that of 17S rRNA (ref. 4 and R. C. v. d. B., J. Retel and R. J. P., manuscript in preparation), it seems reasonable to assume that the 26S rRNA comprises two segments, each showing some similarity to a 17S rRNA molecule. But although competition in hybridization with DNA does mean that the two RNA species exhibit some primary structural homology, it certainly does not indicate that they have identical base sequences.

The number of copies of the ribosomal RNA genes varies considerably between the different plant species studied. It does seem, however, that there is no gross amplification or deletion of these genes during the development of a particular plant.