Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel.

The polypeptide exit tunnel is an important functional compartment of the ribosome where the newly synthesized proteins are surveyed. The tunnel is the target of clinically important macrolide antibiotics. Macrolides plug the tunnel and are believed to stop production of all proteins. Contrary to this view, we show that drug-bound ribosomes can synthesize a distinct subset of cellular polypeptides. The structure of a protein defines its ability to thread through the antibiotic-obstructed tunnel. Synthesis of certain polypeptides that initially bypass translational arrest can be stopped at later stages of elongation while translation of some proteins goes to completion. Our findings reveal that small-molecule effectors can accentuate the discriminatory properties of the ribosomal exit tunnel and that macrolide antibiotics reshape the cellular proteome rather than block global protein synthesis.

Regulation of gene expression by macrolide-induced ribosomal frameshifting.

The expression of many genes is controlled by upstream ORFs (uORFs). Typically, the progression of the ribosome through a regulatory uORF, which depends on the physiological state of the cell, influences the expression of the downstream gene. In the classic mechanism of induction of macrolide resistance genes, antibiotics promote translation arrest within the uORF, and the static ribosome induces a conformational change in mRNA, resulting in the activation of translation of the resistance cistron. We show that ketolide antibiotics, which do not induce ribosome stalling at the uORF of the ermC resistance gene, trigger its expression via a unique mechanism. Ketolides promote frameshifting at the uORF, allowing the translating ribosome to invade the intergenic spacer. The dynamic unfolding of the mRNA structure leads to the activation of resistance. Conceptually similar mechanisms may control other cellular genes. The identified property of ketolides to reduce the fidelity of reading frame maintenance may have medical implications.

Gross Chromosomal Rearrangements in Kluyveromyces marxianus Revealed by Illumina and Oxford Nanopore Sequencing.

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research.

Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center.

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

The general mode of translation inhibition by macrolide antibiotics.

Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. To understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5'-proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The problematic sequence motifs are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segment that contacts the antibiotic molecule in the exit tunnel. Therefore, it appears that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not functioning during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics.

The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide.

The ribosome is able to monitor the structure of the nascent peptide and can stall in response to specific peptide sequences. Such programmed stalling is used for the regulation of gene expression. The molecular mechanisms of the nascent-peptide recognition and ribosome stalling are unknown. We identified the conserved and posttranscriptionally modified 23S rRNA nucleotide m(2)A2503 located at the entrance of the ribosome exit tunnel as a key component of the ribosomal response mechanism. A2503 mutations abolish nascent-peptide-dependent stalling at the leader cistrons of several inducible antibiotic resistance genes and at the secM regulatory gene. Remarkably, lack of the C2 methylation of A2503 significantly function induction of expression of the ermC gene, indicating that the functional role of posttranscriptional modification is to fine-tune ribosome-nascent peptide interactions. Structural and biochemical evidence suggest that m(2)A2503 may act in concert with the previously identified nascent-peptide sensor, A2062, in the ribosome exit tunnel to relay the stalling signal to the peptidyl transferase centre.

Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica.

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3' flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty ("green animals") and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

Digital-to-biological converter for on-demand production of biologics.

Manufacturing processes for biological molecules in the research laboratory have failed to keep pace with the rapid advances in automization and parellelization. We report the development of a digital-to-biological converter for fully automated, versatile and demand-based production of functional biologics starting from DNA sequence information. Specifically, DNA templates, RNA molecules, proteins and viral particles were produced in an automated fashion from digitally transmitted DNA sequences without human intervention.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.

Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life.

Design and synthesis of a minimal bacterial genome.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Macrolide antibiotics in the ribosome exit tunnel: species-specific binding and action.

Macrolide antibiotics bind in the nascent peptide exit tunnel of the ribosome and inhibit protein synthesis. The majority of information on the principles of binding and action of these antibiotics comes from studies that employed model organisms. However, there is a growing understanding that the binding of macrolides to their target, as well as the mode of inhibition of translation, can be strongly influenced by variations in ribosome structure between bacterial species. Awareness of the existence of species-specific differences in drug action and appreciation of the extent of these differences can stimulate future work on developing better macrolide drugs. In this review, representative cases illustrating the organism-specific binding and action of macrolide antibiotics, as well as species-specific mechanisms of resistance are analyzed.

The American Orthopaedic Association's "own the bone" initiative to prevent secondary fractures.

BACKGROUND: The American Orthopaedic Association initiated its Own the Bone pilot project in 2005 in order (1) to assess current orthopaedic practices for the prevention of secondary fractures in adult patients who have sustained a low-energy fracture (fragility fracture), (2) to pilot quality-improvement tools designed to improve the application of evidence-based strategies for the prevention of secondary fractures, and (3) to identify barriers to the broader implementation of the Own the Bone project and explore how to overcome them. METHODS: The ten-month pilot project took place at fourteen sites (thirteen inpatient sites and one outpatient site) and involved 635 participants with a median age of seventy-seven years. The primary outcome measures were the percentages of patients who received (1) counseling on calcium and vitamin-D supplementation, weight-bearing exercise, smoking cessation, and fall prevention, (2) bone mineral density testing, and (3) pharmaceutical intervention to prevent or treat osteoporosis. Secondary outcome measures focused on improved information flow and included the percentage of patients whose physicians were sent a letter recommending the evaluation and treatment of the fracture and the percentage of patients who received a letter recommending that they see their primary-care physician for evaluation and treatment of osteoporosis associated with the fracture. RESULTS: The intervention produced significant improvements (p < 0.0001) in patient counseling on calcium and vitamin-D supplementation, exercise, fall prevention, and communication with primary-care providers and the patients themselves. No improvements were shown in the ordering of bone mineral density testing or the prescription of pharmacotherapy. The most significant improvements (p < 0.0001) were in improved communication with primary-care physicians and in efforts to educate patients about their risk of future fracture. CONCLUSIONS: The Own the Bone initiative offers tools to improve the prevention of secondary fractures and a structure to monitor physician compliance. The American Orthopaedic Association plans to use these quality-improvement tools to stimulate change in both physician and patient behavior following low-energy fractures.