23S rRNA甲基转移酶RrmJ促进细菌50S亚基后期组装

核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控,需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶,能够甲基化修饰核糖体上肽基转移酶中心（peptidyl transferase center, PTC）内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积,因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明,回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善,50S前体累积现象也得到有效缓解。通过共沉淀实验证明,RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S;当加入S-腺苷甲硫氨酸时,该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明,缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明,U2552位点的修饰发生在50S亚基组装晚期特定阶段,这一事件不仅会加速A环的RNA螺旋折叠,另有可能促进附近PTC区域结构成熟。     

Ribosome origins: The relative age of 23S rRNA Domains

The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.     

Gateway Role for rRNA Precursors in Ribosome Assembly

In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.     

The Effect of Ribosome Assembly Cofactors on In Vitro 30S Subunit Reconstitution

Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimP enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimM caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis.     

Mutations in the Escherichia coli23S rRNA Increase the Rate of Peptidyl-tRNA Dissociation from the Ribosome

We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Ribosome Assembly Factors Pwp1 and Nop12 Are Important for Folding of 5.8S rRNA during Ribosome Biogenesis in Saccharomyces cerevisiae

Previous work from our lab suggests that a group of interdependent assembly factors (A₃ factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A₃ factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA₃ pre-rRNA processing, even though the A₃ factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A₃ factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Thiostrepton, an Inhibitor of 50S Ribosome Subunit Function

Thiostrepton inhibits (14)C-leucine incorporation by intact cells of Bacillus megaterium as well as (14)C-phenylalanine incorporation by a poly U-directed extract of Escherichia coli. Extracts of E. coli which are pretreated by incubation with thiostrepton cannot be reactivated by dialysis to more than 5% of their former activity. The 50S ribosome subunit appears to be the site of thiostrepton action, since protein-synthesizing activity can be restored to dialyzed pretreated extracts by supplementation with 50S ribosome subunits but not with 30S ribosome subunits. This technique also provides a simple sensitive method for detection of the biological activity of very small amounts of 50S ribosome subunits.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene.

Characterization of the rRNA operon from the obligate intracellular bacterium Coxiella burnetii has determined the order of the rRNA genes to be 16S-23S-5S. A 444-bp intervening sequence (IVS) was identified to interrupt the 23S rRNA gene beginning at position 1176. The IVS is predicted to form a stem-loop structure formed by flanking inverted repeats, and the absence of intact 23S rRNA molecules suggests that the loop is removed. An open reading frame in the IVS has been identified that shows 70% similarity at the amino acid level to IVS open reading frames characterized from four species of Leptospira.     

Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

Background

YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.

Methodology/Principal Findings

MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only ∼25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix α2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.

Conclusions/Significance

An uncommon means to achieve GTP hydrolysis utilizing a K⁺ ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K⁺ driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases.     

rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.     

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.