Identification of potential allosteric communication pathways between functional sites of the bacterial ribosome by graph and elastic network models

BackgroundAccumulated evidence indicates that bacterial ribosome employs allostery throughout its structure for protein synthesis. The nature of the allosteric communication between remote functional sites remains unclear, but the contact topology and dynamics of residues may play role in transmission of a perturbation to distant sites.Methods/resultsWe employ two computationally efficient approaches – graph and elastic network modeling to gain insights about the allosteric communication in ribosome. Using graph representation of the structure, we perform k-shortest pathways analysis between peptidyl transferase center-ribosomal tunnel, decoding center-peptidyl transferase center - previously reported functional sites having allosteric communication. Detailed analysis on intact structures points to common and alternative shortest pathways preferred by different states of translation. All shortest pathways capture drug target sites and allosterically important regions. Elastic network model further reveals that residues along all pathways have the ability of quickly establishing pair-wise communication and to help the propagation of a perturbation in long-ranges during functional motions of the complex.ConclusionsContact topology and inherent dynamics of ribosome configure potential communication pathways between functional sites in different translation states. Inter-subunit bridges B2a, B3 and P-tRNA come forward for their high potential in assisting allostery during translation. Especially B3 emerges as a potential druggable site.General significanceThis study indicates that the ribosome topology forms a basis for allosteric communication, which can be disrupted by novel drugs to kill drug-resistant bacteria. Our computationally efficient approach not only overlaps with experimental evidence on allosteric regulation in ribosome but also proposes new druggable sites.     

Biophysical simulations and structure-based modeling of residue interaction networks in the tumor suppressor proteins reveal functional role of cancer mutation hotspots in molecular communication

In the current study, we have combined molecular simulations and energetic analysis with dynamics-based network modeling and perturbation response scanning to determine molecular signatures of mutational hotspot residues in the p53, PTEN, and SMAD4 tumor suppressor proteins. By examining structure, energetics and dynamics of these proteins, we have shown that inactivating mutations preferentially target a group of structurally stable residues that play a fundamental role in global propagation of dynamic fluctuations and mediating allosteric interaction networks. Through integration of long-range perturbation dynamics and network-based approaches, we have quantified allosteric potential of residues in the studied proteins. The results have revealed that mutational hotspot sites often correspond to high centrality mediating centers of the residue interaction networks that are responsible for coordination of global dynamic changes and allosteric signaling. Our findings have also suggested that structurally stable mutational hotpots can act as major effectors of allosteric interactions and mutations in these positions are typically associated with severe phenotype. Modeling of shortest inter-residue pathways has shown that mutational hotspot sites can also serve as key mediating bridges of allosteric communication in the p53 and PTEN protein structures. Multiple regression models have indicated that functional significance of mutational hotspots can be strongly associated with the network signatures serving as robust predictors of critical regulatory positions responsible for loss-of-function phenotype. The results of this computational investigation are compared with the experimental studies and reveal molecular signatures of mutational hotspots, providing a plausible rationale for explaining and localizing disease-causing mutations in tumor suppressor genes.     

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides

Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.     

Twist-joints and double twist-joints in RNA structure

Analysis of available RNA crystal structures has allowed us to identify a new family of RNA arrangements that we call double twist-joints, or DTJs. Each DTJ is composed of a double helix that contains two bulges incorporated into different strands and separated from each other by 2 or 3 bp. At each bulge, the double helix is over-twisted, while the unpaired nucleotides of both bulges form a complex network of stacking and hydrogen-bonding with nucleotides of helical regions. In total, we identified 14 DTJ cases, which can be combined in three groups based on common structural characteristics. One DTJ is found in a functional center of the ribosome, another DTJ mediates binding of the pre-tRNA to the RNase P, and two more DTJs form the sensing domains in the glycine riboswitch.     

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.     

Conserved nucleotides within the J domain of the encephalomyocarditis virus internal ribosome entry site are required for activity and for interaction with eIF4G

The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.     

Interpretation of Genomic Variants Using a Unified Biological Network Approach

The decreasing cost of sequencing is leading to a growing repertoire of personal genomes. However, we are lagging behind in understanding the functional consequences of the millions of variants obtained from sequencing. Global system-wide effects of variants in coding genes are particularly poorly understood. It is known that while variants in some genes can lead to diseases, complete disruption of other genes, called ‘loss-of-function tolerant’, is possible with no obvious effect. Here, we build a systems-based classifier to quantitatively estimate the global perturbation caused by deleterious mutations in each gene. We first survey the degree to which gene centrality in various individual networks and a unified ‘Multinet’ correlates with the tolerance to loss-of-function mutations and evolutionary conservation. We find that functionally significant and highly conserved genes tend to be more central in physical protein-protein and regulatory networks. However, this is not the case for metabolic pathways, where the highly central genes have more duplicated copies and are more tolerant to loss-of-function mutations. Integration of three-dimensional protein structures reveals that the correlation with centrality in the protein-protein interaction network is also seen in terms of the number of interaction interfaces used. Finally, combining all the network and evolutionary properties allows us to build a classifier distinguishing functionally essential and loss-of-function tolerant genes with higher accuracy (AUC = 0.91) than any individual property. Application of the classifier to the whole genome shows its strong potential for interpretation of variants involved in Mendelian diseases and in complex disorders probed by genome-wide association studies.     

Potential new antibiotic sites in the ribosome revealed by deleterious mutations in RNA of the large ribosomal subunit

The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.     

Functional epitopes at the ribosome subunit interface

The ribosome is a 2.5-MDa molecular machine that synthesizes cellular proteins encoded in mRNAs. The 30S and 50S subunits of the ribosome associate through structurally defined intersubunit bridges burying 6,000 A(2), 80% of which is buried in conserved RNA-RNA interactions. Intersubunit bridges bind translation factors, may coordinate peptide bond formation and translocation and may be actively remodeled in the post-termination complex, but the functional importance of numerous 30S bridge nucleotides had been unknown. We carried out large-scale combinatorial mutagenesis and in vivo selections on 30S nucleotides that form RNA-RNA intersubunit bridges in the Escherichia coli ribosome. We determined the covariation and functional importance of bridge nucleotides, allowing comparison of the structural interface and phylogenetic data to the functional epitope. Our results reveal how information for ribosome function is partitioned across bridges, and suggest a subset of nucleotides that may have measurable effects on individual steps of the translational cycle.     

Specialized ribosomes: a new frontier in gene regulation and organismal biology

Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than intrinsic regulatory capacity in mRNA translation. However, emerging studies reveal that ribosome activity may be highly regulated. Heterogeneity in ribosome composition resulting from differential expression and post-translational modifications of ribosomal proteins, ribosomal RNA (rRNA) diversity and the activity of ribosome-associated factors may generate 'specialized ribosomes' that have a substantial impact on how the genomic template is translated into functional proteins. Moreover, constitutive components of the ribosome may also exert more specialized activities by virtue of their interactions with specific mRNA regulatory elements such as internal ribosome entry sites (IRESs) or upstream open reading frames (uORFs). Here we discuss the hypothesis that intrinsic regulation by the ribosome acts to selectively translate subsets of mRNAs harbouring unique cis-regulatory elements, thereby introducing an additional level of regulation in gene expression and the life of an organism.     

Structural analysis of kasugamycin inhibition of translation

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).     

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.     

Mutational analysis of an inherently defective translation initiation site.

In a reverse of many studies of translational initiation sites, we have explored the basis for the inactivity of an apparently defective initiation site. Gene VII of the filamentous phage f1 has a translational start site with highly unusual functional properties and a sequence dissimilar to a prokaryotic ribosome binding site. The VII site shows no activity in assays of independent initiation, even in a deletion series designed to remove potentially interfering RNA secondary structure. Activity from the VII site is only observed if the site is coupled to a source of translation immediately upstream, but its efficiency is low at a one-nucleotide spacing from the stop codon of the upstream cistron and extremely sensitive to the distance between the stop codon and the gene VII AUG. These and other atypical characteristics of coupling distinguish the VII site from most coupled initiation sites. To identify the pattern of nucleotide substitutions that give the VII site the capacity for independent initiation, a series of designed and random point mutations were introduced in the sequence. Improving the Shine-Dalgarno complementarity from GG to GGAG or GGAGG made activity detectable, but at only low levels. Random substitutions, each increasing activity above background by a small increment, were found at 16 positions throughout the region of ribosome contact. These substitutions lengthened the Shine-Dalgarno complementarity or changed the G and C residues present in the wild-type site to A or T. Significant activity was not observed unless a strong Shine-Dalgarno sequence and a number of the up-mutations were present together. The nature and distribution of the substitutions and their agreement with the known preferences for nucleotides in initiation sites provide evidence that the VII site's major defect is its primary sequence overall. It appears to lack the specialized sequence required to bind free 30 S ribosomes, and thus depends on the translational coupling process to give it limited activity.     

The translation system of mammalian mitochondria

Oligoribonucleotides and mRNA were used to define properties of the bovine mitoribosomal mRNA binding site. The RNA binding domain on the 28 S subunit spans approx. 80 nucleotides of the template, based on ribosome protection experiments, but the major interaction with the ribosome occurs over a 30 nucleotide stretch. The binding site for E. coli IF3 is conserved in bovine mitoribosomes, but mitochondrial factors appear essential for proper interaction of mRNA with mitoribosomes. The small subunit of bovine mitoribosomes contains a high-affinity binding site for guanyl nucleotides, further indication of specialized mechanisms for initiation complex formation and function of mammalian mitochondrial ribosomes.     

Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.     

Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.     

Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly

Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.