Ribosomal RNA synthesis during cleavage of Ascaris lumbricoides eggs

The appearance of well-defined nucleoli in 4-celled Ascaris embryos is associated with the synthesis of ribosomal RNA.     

Chromosomal and nucleolar RNA synthesis in root tips during mitosis

Comparative rates of RNA synthesis in chromatin and nucleolar fractions during mitosis in root-tip cells of Allium and Nigella were studied by pulse-labeling of cells with tritiated cytidine. Although the rate of RNA synthesis decreases in the condensing chromosomes during prophase, it remains normal in the nucleolar fraction as long as nucleoli are maintained. RNA synthesis stops in mitotic cells lacking distinct nucleoli. In the late telophase or very early interphase cells, RNA synthesis resumes at a faster rate in the pronucleolar bodies than in the chromatin.     

Ribosomal RNA synthesis and processing in a particulate site in the HeLa cell nucleus

A particulate fraction has been isolated from detergent-prepared HeLa cell nuclei. The fraction consists largely of organelles that resemble the nucleoli of intact cells. The 45S RNA that is precursor to 28S and 18S ribosomal RNA is associated with the fraction. The 32S RNA that is labeled after the 45S RNA and is the apparent precursor to 28S RNA is also associated with the fraction. The nucleoplasm contains 28S RNA that behaves as an intermediate between the 32S nucleolar RNA and the 28S cytoplasmic RNA.     

A computational screen for methylation guide snoRNAs in yeast

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Many of the genes for this snoRNA family have remained unidentified in Saccharomyces cerevisiae, despite the availability of a complete genome sequence. Probabilistic modeling methods akin to those used in speech recognition and computational linguistics were used to computationally screen the yeast genome and identify 22 methylation guide snoRNAs, snR50 to snR71. Gene disruptions and other experimental characterization confirmed their methylation guide function. In total, 51 of the 55 ribose methylated sites in yeast ribosomal RNA were assigned to 41 different guide snoRNAs.     

RIBOSOMAL RNA IN THE DEVELOPING CHICK EMBRYO

Developing chick embryos from the stage of primitive-streak formation make ribosomal (28S and 16S) RNA. The size and composition of the RNA appears to be constant throughout the first 7 days of development.     

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.     

Effect of the Vinca alkaloids on RNA synthesis in human cells in vitro

The Vinca alkaloids vinblastine sulfate and vincrystine sulfate, which are mitotic poisons, inhibit RNA synthesis in human (HEp-2) cells cultured in vitro. Analyses of RNA synthesis by cells treated with these drugs by acrylamide gel electrophoresis show that 28s rRNA and to a lesser extent 18s rRNA are preferentially inhibited. The synthesis of tRNA is affected much less than that of rRNA. The present experiments suggest that the drugs inhibit both the synthesis and processing of the nucleolar RNA precursors of rRNA. An explanation is also given for previous reports that these alkaloids preferentially inhibit the synthesis of tRNA in animal cells in vitro.     

Polypeptide synthesis in sea urchin embryogenesis: an examination with synthetic polyribonucleotides

After fertilization of the sea urchin egg the rate of protein synthesis by a cell-free ribosomal system increases markedly. This increase can be attributed to newly synthesized messenger RNA, since (i) the rate of polypeptide synthesis elicited by synthetic messenger polyribonucleotides changes only slightly after fertilization, and (ii) the enzymes for the formation of amino acyl transfer RNA's of phenylalanine and leucine and the polymerization of polypeptide are in excess in the unfertilized egg. Polypeptide synthesis has been characterized in development to the gastrula stage.     

Regulation of bacterial growth

Is the control of bacterial metabolism so complex? The answer can be found in a simple experiment. Two cultures of bacteria are grown in different mediums. One contains as the carbon and nitrogen sources a mixture of amino acids, while the other contains only glucose and ammonia, so that the cells must synthesize all of the amino acids. The results show that insofar as the cells in both cultures grow at comparable rates, they will have the same composition in terms of DNA, RNA, and protein (30). To explain this phenomena I have argued that through the control mechanisms responsible for the distribution of substrates in intermediary metabolism, the substrates of protein synthesis are produced at concentrations and rates commensurate with the ability of the environment to support growth. The provision of these substrates relative to the ability of the protein forming system to utilize them regulates the synthesis of ribosomal and transfer RNA, which, after adjustment for various modulating influences, such as nonfunctioning ribosomes or ribosomal RNA turnover, brings the number of functioning ribosomes to a point in keeping with the provision of external nutrients. The synthesis of messenger (or total) RNA, ribosomal proteins, and DNA, and the process of cell division, for example, are subject to their own controls, but through the burden they each place on intermediary metabolism, they provide a means for partitioning the cell's metabolic resources. It might be noted that this view may not be very far from the idea once held that the rate at which each of the transfer RNA's was changed by amino acids regulate the synthesis of bacterial RNA, but growth regulation is clearly more complicated than implied by that model (76).     

Actinomycin D; renewed RNA synthesis after removal from mammalian cells

Suppressed RNA synthesis exceeded control rates 2 hours after removal of actinomycin from serial lines of green monkey kidney cells in exponentially dividing monolayer cultures. Enhanced synthesis was directly related to the dose of actinomycin, and release from inhibition was temperature dependent. A broad range of RNA sizes was made after removal of drug, but cytoplasmic and nucleolar sedimentation profiles were abnormal for at least 20 hours.     

Turnover of ribosomal RNA in rat liver

After a single injection of radioactive orotic acid and a "chase" of nonradioactive precursor, the specific activity of ribosomal RNA in rat liver decreases logarithmically at a rate corresponding to a half-life of about S days. The possible significance of this result is discussed with regard to control of protein synthesis.     

Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification

Mutations in DKC1 cause dyskeratosis congenita (DC), a disease characterized by premature aging and increased tumor susceptibility. The DKC1 protein binds to the box H + ACA small nucleolar RNAs and the RNA component of telomerase. Here we show that hypomorphic Dkc1 mutant (Dkc1m) mice recapitulate in the first and second generations (G1 and G2) the clinical features of DC. Dkc1m cells from G1 and G2 mice were impaired in ribosomal RNA pseudouridylation before the onset of disease. Reductions of telomere length in Dkc1m mice became evident only in later generations. These results suggest that deregulated ribosome function is important in the initiation of DC, whereas telomere shortening may modify and/or exacerbate DC.     

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.     

Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1

Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.     

Involvement of RNA in the synthesis of proteins

We can now have considerable confidence that the broad features of protein synthesis are understood. The involvement of RNA is very much more complicated than was imagined in 1953. There is not one functional RNA. Instead, protein synthesis demands the ordered interaction of three classes of RNA-ribosomal, soluble, and messenger. Many important questions, however, remain unanswered. For instance, there is no theoretical framework for the ribosomal subunits, nor for that matter, do we understand the functional significance of ribosomal RNA. Most satisfying is the realization that all the steps in protein replication will be shown to involve well-understood chemical forces. As yet we do not know all the details. For example, are the DNA base pairs involved in messenger RNA selection of the corresponding amino-acyl-sRNA? With luck, this will soon be known. We can thus have every expectation that future progress in understanding selective protein synthesis (and its consequences for embryology) will have a similarly well-defined and, when understood, easy-to-comprehend chemical basis (62).     

Structural biology. The ribosome is a ribozyme

Ribosomes, the cellular factories that manufacture proteins, contain both RNA and protein, but exactly how all of the different ribosomal components contribute to protein synthesis is still not clear. Now, as Thomas Cech explains in his Perspective, atomic resolution of the structure of the large ribosomal subunit reveals that, as predicted by those convinced of a prebiotic RNA world, RNA is the catalytic component with proteins being the structural units that support and stabilize it (Ban et al., Nissen et al., Muth et al.).     

High macromolecular synthesis with low metabolic cost in Antarctic sea urchin embryos

Assessing the energy costs of development in extreme environments is important for understanding how organisms can exist at the margins of the biosphere. Macromolecular turnover rates of RNA and protein were measured at -1.5 degrees C during early development of an Antarctic sea urchin. Contrary to expectations of low synthesis with low metabolism at low temperatures, protein and RNA synthesis rates exhibited temperature compensation and were equivalent to rates in temperate sea urchin embryos. High protein metabolism with a low metabolic rate is energetically possible in this Antarctic sea urchin because the energy cost of protein turnover, 0.45 joules per milligram of protein, is 1/25th the values reported for other animals.     

Crystal structure of an initiation factor bound to the 30S ribosomal subunit

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.