Structural Transitions in Ribonucleic Acid During Ribosome Development

Amino acid deprivation of a "relaxed" auxotroph of Escherichia coli results in the accumulation of protein-deficient, immature ribosomes ("relaxed particles"). The ribonucleic acid (RNA) of these particles was shown to differ from mature ribosomal RNA in both sedimentation characteristics and in elution from columns of methylated albumin-keiselguhr. When relaxed particles were allowed to become converted to mature ribosomes, the unique properties of the RNA were lost, and this RNA became indistinguishable from mature RNA. The conversion of relaxed particles to ribosomes did not involve degradation and resynthesis of RNA. It is concluded that ribosomal RNA undergoes a configurational transition during ribosome development, and that this transition is not the result of changes in the primary structure of the RNA.     

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.     

Effect of ermC leader region mutations on induced mRNA stability.

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.     

Ribosome Development and the Methylation of Ribosomal Ribonucleic Acid

Immature ribonucleoprotein particles accumulate in amino acid-starved cells of a relaxed mutant of Escherichia coli. The ribonucleic acid (RNA) of these particles is nonmethylated in cells starved for methionine. However, in bacteria starved for arginine, lysine, or histidine, the RNA of these particles is one-half methylated. The relationship of submethylation to a structural alteration in the same RNA was studied. The results of kinetic studies showed that submethylation and the structural transition are not causally related, since they are described by different rate constants. Moreover, it was possible to accumulate fully methylated immature-particle RNA that possessed the sedimentation and chromatographic properties of nonmethylated RNA. It was concluded that, during the normal course of ribosome development, methylation of ribosomal RNA is completed prior to the final maturation steps.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

微生物核糖体工程研究进展

摘要：微生物获得特定类型的抗性突变,不仅反映了其核糖体或RNA多聚酶上相关靶位点结构的改变,也对突变菌株次级代谢产物（抗生素等）的生物合成能力产生深刻影响,因此筛选抗性突变株可作为微生物推理选育的途径之一。“核糖体工程”是利用微生物的各类抗性突变为筛选标记,高效获得次生代谢产物合成能力提高的突变株的推理育种新方法。本文综述了微生物“核糖体工程”的概念、各类突变的作用机理,并着重介绍组合抗性突变在提高出发菌株次级代谢产物产量方面的应用。     

Ribonucleic Acid Regulation in Amino Acid-Limited Cultures of Escherichia coli Grown in a Chemostat

The regulation of ribonucleic acid (RNA) synthesis was examined in cultures of bacteria whose growth was limited in the chemostat by the supply of a required amino acid. Strains possessing the relaxed (relA) mutation accumulated excess RNA (relative to protein) at low growth rates when growth was limited by arginine, histidine, or cysteine but not when limited by methionine. In contrast, stringent (relA(+)) strains maintained a constant RNA/protein ratio with decreasing growth rate regardless of the amino acid used to limit growth. The presence of excess RNA in relaxed strains was accompanied by an absence of increase in RNA production upon addition of chloramphenicol, a lag upon shift-up in growth by addition of excess of the limiting amino acid, and a decreased rate of production of beta-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50S and 30S ribosomal subunits. Microscope examination of the relaxed strains during histidine-, arginine-, or cysteine-limited growth in the chemostat showed them to be 10 to 20 times longer in size than the stringent strains. Also, cell density was reduced to one-tenth when the increased size was observed. An analysis of the amount of ppGpp present in all slow-growing amino acid-limited cultures (relaxed and stringent) demonstrated that only basal levels of ppGpp were made. These data are consistent with the hypothesis that when growth is limited in the chemostat by an initiation event in protein synthesis, i.e., limited methionine, RNA regulation occurs in relaxed as well as stringent strains. Also, when other amino acids are limiting in concentration during translation, errors occur in relaxed strains, resulting in misread proteins.     

A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and -1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.     

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.     

The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo

Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB , G operon that prevents synthesis of ribosomal proteins L28 and L33. The mutation was lethal unless synthesis of protein L28 was induced from a plasmid. Without protein L28, RNA and protein synthesis were linear rather than exponential. No 70S ribosomes were made. Instead, RNA accumulated in '30S material' and '47S particles'; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins. When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild-type control. Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.     

Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA.     

Cohesion promotes nucleolar structure and function

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.     

An RNA-dependent UMP-incorporating activity is associated with the small subunit of cytoplasmic ribosomes in bloodstream forms of Trypanosoma brucei.

Trypanosoma brucei cytoplasm contained a UMP-incorporating activity which was dependent upon the presence of endogenous RNA, stimulated by exogenous RNA and precipitable by ammonium sulphate. The activity cosedimented with ribosomes, and after ribosome dissociation using EDTA was mainly associated with the small (31S) subunit. Ribosome-associated activity was selective for UTP as a substrate and greatly stimulated by the presence of at least one other nucleoside triphosphate.     

Cap-dependent translation without base-by-base scanning of an messenger ribonucleic acid

'Ribosome scanning' is the generally accepted mechanism for explaining how a ribosome finds an initiation codon located far removed from the ribosome recruiting site (cap structure). However, the molecular characteristics of ribosome scanning along 5' untranslated regions (UTRs) remain obscure. Herein, using a rabbit reticulocyte lysate (RRL) system and artificial ribonucleic acid (RNA) constructs composed of a capped leader RNA and an uncapped reporter RNA annealed through a double-stranded RNA (dsRNA) bridge, we show that the ribosome can efficiently bypass a stable, dsRNA region without melting the structure. The insertion of an upstream open reading frame in the capped leader RNA impaired the translation of reporter RNA, indicating that a ribosome associated with the 5'-end explores the regions upstream of the dsRNA bridge in search of the initiation codon. These data indicate that a ribosome may skip part(s) of an messenger RNA 5'UTR without thoroughly scanning it.     

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.     

Identification of a structural motif of 23S rRNA interacting with 5S rRNA.

To identify RNA motifs interacting with 5S rRNA, a systematic evolution of ligands by exponential enrichment experiment was applied. Some of the resulting RNA aptamers contained a consensus sequence similar to the sequence in the loop region of helix 89 of 23S rRNA. We show that the synthetic helix 89 RNA motif indeed interacted with 5S rRNA and that the region around loop B of 5S rRNA was involved in this interaction. These results suggest the presence of a novel RNA-RNA interaction between 23S rRNA and 5S rRNA which may play an important role in the ribosome function.     

The distribution of ribosomal ribonucleic acids among subcellular fractions from bacteria and the adverse effect of the membrane fraction on the stability of ribosomes

1. The distributions of nucleic acids and protein among fractions obtained by differential centrifugation from species of Pseudomonas, Aerobacter, Escherichia, Proteus and Bacillus have been studied. 2. The DNA in a cell wall-membrane fraction obtained by low-speed centrifugation from the Gram-negative species could be removed by homogenizing and subsequent washing. About 7-14% of the total RNA remained firmly attached and resembled ribosomal RNA in base composition. A similar fraction from the Gram-positive B. subtilis contained about one-half of the total bacterial DNA and only 60% of this could be removed by homogenizing and subsequent washing. 3. A deposit obtained by high-speed centrifugation could be separated into a heavy ribosome layer and a light turbid layer. In E. coli B the latter contained about equal concentrations of RNA and DNA and accounted for about one-half of the total bacterial NADP-activated 6-phosphogluconate dehydrogenase. 4. The washed cell wall-membrane fraction from most species accelerated the degradation of ribosomes. In Pr. vulgaris the activity of this fraction was exceptionally high and resulted in the progressive degradation of ribosomes during their isolation from this species. 5. A possible connexion between ribosome degradation and the synthesis of flagella is discussed in the light of these results.