Selection for intragenic suppressors of lethal 23S rRNA mutations in Escherichia coli identifies residues important for ribosome assembly and function

Mutations in several functionally important regions of the 23S rRNA of E. coli increase the levels of frameshifting and readthrough of stop codons. These mutations include U2555A, U2555G, ΔA1916 and U2493C. The mutant rRNAs are lethal when expressed at high levels from a plasmid, in strains also expressing wild type rRNA from chromosomal rrn operons. The lethal phenotype can be suppressed by a range of second-site mutations in 23S rRNA. However, analysis of the functionality of the double mutant rRNAs in heterogeneous ribosome populations shows that in general, the second site mutations do not restore function. Instead, they prevent the assembly, or entry of the mutant 50S subunits into the functioning 70S ribosome and polysome pools, by affecting the competitiveness of the mutant subunits for association with 30S particles. The second-site mutations lie in regions of the 23S rRNA involved in subunit assembly, intersubunit bridge formation and interactions of the ribosome with tRNAs and factors. These second site suppressor mutations thus define functionally important rRNA nucleotides and this approach may be of general use in the functional mapping of large RNAs.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ†

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature‐dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′‐terminus, and that the 3′‐terminus is only trimmed at a later step. At normal temperatures (30°C?37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse‐chase analysis. Assembly defects are partially compensated by an overexpression of other heat‐shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid‐driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis.     

Cracking pre‐40S ribosomal subunit structure by systematic analyses of RNA–protein cross‐linking

Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre‐ribosomal particles. We report rRNA‐binding sites for six late‐acting 40S ribosome synthesis factors, three of which cluster around the 3′ end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ structure formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1‐ and Tsr1‐binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3′‐endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre‐rRNA. Nob1 binds before pre‐rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.     

Reconstitution of 50 S ribosomal subunits from Bacillus stearothermophilus with 5 S RNA from spinach chloroplasts and low-Mr RNA from mitochondria of Locusta migratoria and bovine liver

Reconstitution experiments with 50 S ribosomal subunits from Bacillus stearothermophilus demonstrate that spinach chloroplast 5 S rRNA can be incorporated into the bacterial ribosome and yield biologically active particles, thereby establishing the eubacterial nature of chloroplast 5 S rRNA. In contrast, mitochondria from Locusta migratoria or bovine liver do not appear to contain discrete, low-Mr RNAs, which can replace 5 S rRNA in the functional reconstitution of B. stearothermophilus ribosomes.     

Use of Lead(II) to Probe the Structure of Large RNA's. Conformation of the 3′ Terminal Domain of E. coli 16S rRNA and its Involvement in Building the tRNA Binding Sites

Abstract

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3′ domain of the E. coli 16S rRNA (nucleotides 1295–1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational ajustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337,1400,1500 and near the 3′ end of the RNA The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399–1400, 1491–1492 and 1505) protections. Athird class ofprotection(1344–1345,1393–1395,1403–1409,1412–1414, 1504, 1506–1507 and 1517–1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.     

The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

Exploring the selectivity of a ligand complex with CDK2/CDK1: a molecular dynamics simulation approach

Cyclin‐dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Their inhibitors have entered in clinical trials to treat cancer. Very recently, Heathcote et al. (J. Med. Chem. 2010, 53:8508–8522) have found a ligand BS194 that has a high affinity with CDK2 (IC₅₀ = 3 nm ) but shows low affinity with CDK1 (IC₅₀ = 30 nm ). To understand the selectivity, we used homology modeling, molecular docking, molecular dynamics, and free‐energy calculation to analyze the interactions. A rational three‐dimensional model of the CDK1/BS194 complex is built. We found that Leu83 is a key residue that recognizes BS194 more effectively with CDK2 with good binding free energies rather than CDK1. Energetic analysis reveals that van der Waals interaction and non‐polar contributions to solvent are favorable in the formation of complexes and amine group of the ligand, which plays a crucial role for binding selectivity between CDK2 and CDK1. Copyright © 2012 John Wiley & Sons, Ltd.     

Role of protein L25 and its contact with protein L16 in maintaining the active state of <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomes <Emphasis Type="Italic">in vivo</Emphasis>

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.     

Role of Escherichia coli YbeY,a highly conserved protein,in rRNA processing

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′‐ and 3′‐ends of 16S rRNA as well as maturation of the 5′‐termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.     

Experimental and molecular docking investigation on DNA interaction of N‐substituted phthalimides: antibacterial,antioxidant and hemolytic activities

A series of Schiff base molecules derived from a phthalimide scaffold was investigated as efficient antibacterial, antioxidant and DNA‐interacting agents. The spectroscopic characterization of these derivatives was studied in detail using elemental analysis and spectroscopic techniques. The DNA‐binding profile of title molecules against Ct‐DNA (calf thymus) was investigated by absorbance, fluorescence, hydrodynamics and thermal denaturation investigations. The bacterial inhibition potential of these molecules was investigated against Escherichia coli and Staphylococcus aureus. Molecule 3c emerged as the most active against S. aureus (IC₅₀: 14.8 μg/mL), whereas compounds 3a and 3b displayed potential antibacterial activities against E. coli (IC₅₀: 49.7 and 67.6 μg/mL). Molecular docking studies of these compounds against GlcN‐6‐P synthase were carried out to rationalize antibacterial efficiency of these molecules. These newly synthesized molecules were screened for their scavenging capacity against 2,2‐diphenyl‐1‐picryl‐hydrazyl (DPPH) and H₂O₂ free radicals and the results were compared with ascorbic acid as synthetic antioxidant. The title molecules 3a, 3b and 3e showed less than 20% hemolysis, which indicated their significant non‐toxic behavior.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Single rRNA Helices Bind Independently to the Protein‐Conducting Channel SecYEG

Ribosomes tightly interact with protein‐conducting channels in the plasma membrane of bacteria (SecYEG) and in the endoplasmic reticulum of eukaryotes (Sec61 complex). This interaction is mediated by multiple junctions and is highly conserved during evolution. Although it is well known that both ribosomal proteins and ribosomal RNA (rRNA) are involved in the ribosome–channel interaction, detailed analyses on how these components contribute to this binding are lacking. Here, we demonstrate that the evolutionary conservation of ribosome binding is solely mediated by rRNA. Moreover, we show that in vitro transcribed 23 S rRNA binds with similar characteristics to protein translocation channels as native 23 S rRNA or 50 S ribosomal subunits. This indicates that base modifications, which exist in native rRNA, do not crucially influence the binding. In two of the ribosome‐channel junctions (c1 and c2), exclusively rRNA helices are involved. Using in vitro transcribed rRNA mutants, we now provide evidence that large parts of the rRNA can be deleted without altering its binding properties, as long as the rRNA helices of the c1 and c2 junctions remain intact. We demonstrate that the connection sites c1 and c2 generate high‐affinity binding sites that act independently of each other. This could explain why membrane‐bound ribosomes have an extremely low off‐rate .     

细菌核糖体的结构和功能

二十多年来,国际上几家实验室独立地竞争性地应用高分辨率X-射线衍射技术在原子水平上绘制出了细菌完整核糖体及其亚基精细的三维结构图,为其生物功能的研究提供了清晰的结构基础。由于这项伟大的科学成果,美国科学家文卡特拉曼·拉马克里希南（Venkatraman Ramakrishnan）、托马斯．施泰茨（Thomas A．Steitz）和以色列女科学家阿达．约纳特（Ada E．Yonath）三人荣获2009年度诺贝尔化学奖。细菌核糖体是细胞中合成蛋白质的一种细胞器,包括大小不同的两个亚基,由3种RNA和50多种不同的蛋白质组成。     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.