Irene S. Gabashvili

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

The Polypeptide Tunnel System in the Ribosome and Its Gating in Erythromycin Resistance Mutants of L4 and L22

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three‐dimensional cryo‐electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912–910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885–887 (GGG) and 888–890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A‐site tRNA‐binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Ion Channel Gene Expression in the Inner Ear

The ion channel genome is still being defined despite numerous publications on the subject. The ion channel transcriptome is even more difficult to assess. Using high-throughput computational tools, we surveyed all available inner ear cDNA libraries to identify genes coding for ion channels. We mapped over 100,000 expressed sequence tags (ESTs) derived from human cochlea, mouse organ of Corti, mouse and zebrafish inner ear, and rat vestibular end organs to Homo sapiens, Mus musculus, Danio rerio, and Rattus norvegicus genomes. A survey of EST data alone reveals that at least a third of the ion channel genome is expressed in the inner ear, with highest expression occurring in hair cell-enriched mouse organ of Corti and rat vestibule. Our data and comparisons with other experimental techniques that measure gene expression show that every method has its limitations and does not per se provide a complete coverage of the inner ear ion channelome. In addition, the data show that most genes produce alternative transcripts with the same spectrum across multiple organisms, no ion channel gene variants are unique to the inner ear, and many splice variants have yet to be annotated. Our high-throughput approach offers a qualitative computational and experimental analysis of ion channel genes in inner ear cDNA collections. A lack of data and incomplete gene annotations prevent both rigorous statistical analyses and comparisons of entire ion channelomes derived from different tissues and organisms.

Dynamics of double stranded DNA reptation from bacteriophage

The dynamics of dsDNA release process from a phage head has been analyzed theoretically. The process was considered as dsDNA reptation through the phage tail. The driving force is assumed to be the decrease of the DNA globule free energy on its releasing from the head in the surrounding medium. The results of the equilibrium theory on an intraphage DNA globule were applied. Three possible sources of friction were examined. The first one is in the inner channel of the tail. The second is the friction of DNA segments in the whole globule volume, which is essential when the globule decondensation is a collective process, at simultaneous moving of all the turns (mechanism 1). The third is the globule friction with the capsid inner surface, that is most important when decondensation proceeds via the globule rotation as a whole spool (mechanism 2). Mechanism 1 would require a lot of time for ejection. Mechanism 2 would lead to different ejection dynamics of short- and long-tailed phages. Comparison of the theoretical results with the published experimental data argues in favor of mechanism 2.

Mining biochemical information: lessons taught by the ribosome.

The publication of the crystal structures of the ribosome offers an opportunity to retrospectively evaluate the information content of hundreds of qualitative biochemical and biophysical studies of these structures. We assessed the correspondence between more than 2,500 experimental proximity measurements and the distances observed in the ribosomal crystals. Although detailed experimental procedures and protocols are unique in almost each analyzed paper, the data can be grouped into subsets with similar patterns and analyzed in an integrative fashion. We found that, for crosslinking, footprinting, and cleavage data, the corresponding distances observed in crystal structures generally did not exceed the maximum values expected (from the estimated length of the agent and maximal anticipated deviations from the conformations found in crystals). However, the distribution of distances had heavier tails than those typically assumed when building three-dimensional models, and the fraction of incompatible distances was greater than expected. Some of these incompatibilities can be attributed to the experimental methods used. In addition, the accuracy of these procedures appears to be sensitive to the different reactivities, flexibilities, and interactions among the components. These findings demonstrate the necessity of a very careful analysis of data used for structural modeling and consideration of all possible parameters that could potentially influence the quality of measurements. We conclude that experimental proximity measurements can provide useful distance information for structural modeling, but with a broad distribution of inferred distance ranges. We also conclude that development of automated modeling approaches would benefit from better annotations of experimental data for detection and interpretation of their significance.

Qualitative models of molecular function: linking genetic polymorphisms of tRNA to their functional sequelae

The exponential growth in the volume of biological information available makes it difficult for researchers to assemble the details into coherent models. Although an accurate model is ideal, full details are not generally available and are gained only incrementally. Therefore, as a first step toward integration of information, we propose a knowledge model for the qualitative representation of the relationships between mutations in genes and their effects at molecular cellular and clinical phenotypic levels. Our framework combines and extends two components: 1) a workflow model that allows hierarchical process and participant specifications; 2) Transparent Access to Multiple Bioinformatics Information Sources and the Unified Medical Language System, which serve as controlled biological and medical terminologies. By mapping our framework to Petri nets, we can perform qualitative simulations to validate models, and aid in predicting system behavior in the presence of dysfunctional components. This can be a step toward accurate quantitative models. Our application domain is the role of transfer ribonucleic acid molecules in protein translation-related disease. As an initial evaluation, we show that Petri nets derived from the historic and current views of the translation process yield different dynamic behavior. Our model is available at http://smi.stanford.edu/projects/helix/pubs/process-model/.

Differential gene expression in the auditory system

Hearing disorders affect over 10% of the population and this ratio is dramatically increasing with age. Development of appropriate therapeutic approaches requires understanding of the auditory system, which remains largely incomplete. We have identified hearing-specific genes and pathways by mapping over 15000 cochlear expressed sequence tags (ESTs) to the human genome (NCBI Build 35) and comparing it to other EST clusters (Unigene Build 183). A number of novel potentially cochlear-specific genes discovered in this work are currently being verified by experimental studies. The software tool developed for this task is based on a fast bidirectional multiple pattern search algorithm. Patterns used for scoring and selection of loci include EST subsequences, cloning-process identifiers, and genomic and external contamination determinants. Comparison of our results with other programs and available annotations shows that the software developed provides potentially the fastest, yet reliable mapping of ESTs.

Community-Led Research Discovers Links Between Elusive Symptoms And Clinical Tests

Human breath and body odors have been used for diagnosis of serious and life-threatening conditions since the dawn of medical practice. More recently, it has been recognized that malodors without accompanying physical symptoms could be a sign of psychologically but not physically debilitating disorders such as Trimethylaminuria (TMAU). Self-reported intermittent odors without apparent cause, are, however, still treated with suspicion by medical professionals. Most cases of socially-disabling idiopathic malodor remain undiagnosed and there are no guidelines for diagnostic tests nor treatment options that extend beyond TMAU. Internationally-recruited volunteers with undiagnosed body odor and halitosis enrolled to participate in our study, registered as NCT02692495 at clinicaltrials.gov. Each volunteer underwent several blood and urine tests conducted by Biolab Medical Unit, a medical referral laboratory in London, specializing in nutritional and environmental medicine. Intestinal permeability measurements were strikingly different for subjects that named the nose/mouth as the malodor source(s) versus other, often unidentified, body regions. Furthermore, metabolite levels in blood and urine allowed matching of participants by dietary sensitivities and the type of odor reported, emphasizing the potential of harnessing patients’ olfactory observations. In discussing the anecdotal “People are Allergic to Me” condition (PATM), we show how it fits into the picture.

EST-based analysis of gene expression in the human cochlea

Hearing is one of the vital senses helping to perceive, reflect and communicate with the world around us. Genetics, developmental conditions, mechanical damage, infections, ototoxic medications, and aging are among the factors disabling or deteriorating this sense. The cochlea is a sensory organ responsible for hearing. Over 15,000 expressed sequence tags (ESTs) extracted from this organ had been previously clustered with other sequences and aligned to earlier versions of the human genome and known genes. This is especially important as more genomic and phenotypic data becomes available almost on a daily basis. Many transcripts corresponding to ESTs present in the dataset might not be expressed as proteins, but instead are degraded by nonsense-mediated mRNA decay or other cell surveillance mechanisms. Large-scale sequencing of tissue-specific genes and fast yet reliable mapping of sequences will help to identify key components of sound transduction and speed up progress in hearing research.