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In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15–18 nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases.

© 2016 Elsevier Inc. The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

Summary In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits—products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally-relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature.

Summary The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo-designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions., Graphical Abstract, Highlights • SNIPRs are riboregulators with single-nucleotide-specific RNA detection capabilities • They operate in live cells and in paper-based cell-free reactions • Automated in silico design allows SNIPRs to detect many different harmful mutations • Isothermal SNIPR tests enable portable human genotyping and viral strain detection, The development of high-specificity RNA toehold-based sensors allows for the discrimination of point mutations and RNA base modifications and enables genotypic diagnosis for E. coli, human samples, and Zika virus strains

Class 1 CRISPR-Cas systems are characterized by effector modules consisting of multiple subunits. Class 1 systems comprise about 90% of all CRISPR-Cas loci identified in bacteria and archaea and can target both DNA and RNA.

Cells respond to their environment by sensing signals and translating them into changes in gene expression. In recent years, synthetic networks have been designed in both prokaryotic and eukaryotic systems to create new functionalities and for specific applications. In this review, we discuss the challenges associated with engineering signal transduction pathways. Furthermore, we address advantages and disadvantages of engineering signaling pathways in prokaryotic and eukaryotic cells, highlighting recent examples, and discuss how progress in synthetic biology might impact biotechnology and biomedicine.

Recent advances have demonstrated that bacterial cells have an exquisitely organized and dynamic subcellular architecture. Like their eukaryotic counterparts, bacteria employ a full complement of cytoskeletal proteins, localize proteins and DNA to specific subcellular addresses at specific times, and use intercellular signaling to coordinate multicellular events. The striking conceptual and molecular similarities between prokaryotic and eukaryotic cell biology thus make bacteria powerful model systems for studying fundamental cellular questions.

GroEL/S chaperonin ring complexes fold many unrelated proteins. To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP). Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle. These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates. This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.

Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in surprisingly similar ways. Here, I examine common modes of maintaining nonrandom gene organization in both prokaryotes and eukaryotes, the different ways these organizations have likely arisen, and classes of organization that may be unique to one group or the other.

The appearance of these structures represents a historic advance in our understanding of one of molecular biology's most remarkable enzymes. Here we have suggested several possible structural bases for key steps in its function. Although speculative, they should serve as stimuli for future work. Hopefully structures of the bacterial RNAP·σ complex, yRNAPII, and a ternary transcription complex that better define the paths of RNA and DNA at resolutions comparable to the Taq RNAP structure will follow soon. These structures should make RNAP an appealing target for rational drug design. We are poised at the advent of an exciting era in which the mechanisms of initiation, elongation, pausing, arrest, and termination can be tested rigorously and finally described in molecular detail.

The replicon model, proposed by Jacob and Brenner (1963) to explain the regulation of prokaryotic DNA replication, has proved remarkably robust. Despite the manyvariations in initiation mechanism discovered since 1963, the replicon model may be applicable, with minor modifications, to regulation of DNA replication in all organisms. For several years, the Replicon Club of Paris, a group of French scientists interested in regulation of replication, has been meeting to discuss new results. This year, with support from the French Centre National de la Recherche Scientifique (CNRS), the Replicon Club sponsored an international Jacques Monod Conference on Prokaryotic and Eukaryotic Replicons. The meeting was organized by G. Buttin (Institut Pasteur, Paris) with assistance from A. Falaschi (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy) and M. Kohiyama (Institut Jacques Monod, Paris) and was held at the CNRS’s Centre Paul Langevin in the Alpine village of Aussois, France, from June 18-22, 1995. There, invigorated by beautiful surroundings and excellent cuisine, scientists from around the world provided evidence both for the vitality of the replicon model and for substantial advances in our understanding of the regulation of replication.

Steve Busby’ and Richard H. Ebrightt ‘School of Biochemistry University of Birmingham Birmingham 615 2lT England TDepartment of Chemistry and Waksman Institute Rutgers University New Brunswick, New Jersey 08855 Escherichia coli RNA polymerase holoenzyme (RNAP) has been the object of intense study since its discovery. RNAP consists of core enzyme, with subunit composition

SummaryThe ribosome is a complex molecular machine that, in order to synthesize proteins, has to decode mRNAs by pairing their codons with matching tRNAs. Decoding is a major determinant of fitness and requires accurate and fast selection of correct tRNAs among many similar competitors. However, it is unclear whether the modern ribosome, and in particular its large conformational changes during decoding, are the outcome of adaptation to its task as a decoder or the result of other constraints. Here, we derive the energy landscape that provides optimal discrimination between competing substrates and thereby optimal tRNA decoding. We show that the measured landscape of the prokaryotic ribosome is sculpted in this way. This model suggests that conformational changes of the ribosome and tRNA during decoding are means to obtain an optimal decoder. Our analysis puts forward a generic mechanism that may be utilized broadly by molecular recognition systems.

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Transcription start site selection in eukaryotes is mediated through combinations of the TATA, initiator (Inr), and downstream promoter elements (DPE). In Trichomonas vaginalis, a parabasalian flagellate thought to represent an ancient eukaryote lineage, the Inr appears to be solely responsible for start site selection and is recognized by the initiator binding protein 39 kDa (IBP39). IBP39 contains an N-terminal Inr binding domain (IBD) connected via a flexible linker to a C-terminal domain (C domain). Here we present crystal structures of the apoIBD and IBD-Inr complexes and the C domain. The IBD structures reveal a winged-helix motif with prokaryotic and eukaryotic features and a scaffold similar to that of ETS-family proteins. The C domain structure and biochemical studies indicate that it interacts with the T. vaginalis RNAP II large subunit C-terminal domain. These data suggest that binding of IBP39 to the Inr directly recruits RNAP II and in this way initiates transcription.

The periplasmic PapD-like chaperones have long been known to be necessary for the assembly of bacterial surface organelles. New structural work now suggests that they control assembly by arresting subunit folding. This step may be required to preserve energy for fiber formation.

Nucleotide excision repair (NER) was discovered in both prokaryotes and eukaryotes in the 1960s (Friedberg et al., 2005). The process corrects a wide spectrum of damage to DNA bases that results in distortions in the native conformation of DNA, including damage induced by ultraviolet (UV) light and by a plethora of chemicals. NER comprises two distinct subpathways. Global genome repair (GGR) repairs lesions in regions of the genome that are transcriptionally silent, and transcription-coupled repair (TCR) repairs lesions in regions of the genome that are transcriptionally active. A key difference between these two NER pathways is the molecular mechanism used to recognize the damaged base (designated by a red star in the SnapShot figure).

The regulatory logic and underlying mechanisms of eukaryotic gene regulation are ideally suited for achieving extremely complex patterns of gene expression. Aside from the inherently combinatorial nature of gene activation, eukaryotic activators and repressors can function indirectly by recruiting chromatin modifying activities and hence creating domains of altered chromatin that vary in extent from individual nucleosomes to large genomic regions. Consequently, a eukaryotic promoter can exist in a variety of stable and intermediate states that are transcriptionally inactive (Figure 2Figure 2); these states correspond to the chromatin modifying activities recruited by particular activators or repressors and hence can be regulated by cell cycle and developmental signals. The initiation, propagation, maintenance, and dissolution of such intermediate states can account for many important phenomena at the level of individual genes: transient initiating events playing a determining role in transcription at later stages of development; epigenetic memory through cell division cycles for maintenance of differentiated states; sequential opening of chromatin structure in complex genetic loci; generation of large-scale heterochromatic regions. It is hard to imagine such phenomena occurring in prokaryotes given that activators and repressors function rather directly on RNA polymerase in the context of a transcriptionally nonrestrictive ground state. Lastly, activator- and repressor-dependent recruitment of chromatin-modifying complexes may explain why regulation of other eukaryotic processes, such as DNA replication and site-specific recombination, is associated with transcriptional regulatory proteins, but not transcription per se.

Rapid protein synthesis in bacteria requires the G proteins IF2, EF-Tu, EF-G, and RF3. These factors catalyze all major steps of mRNA translation in a GTP-dependent manner. Here, it is shown how the position of peptidyl-tRNA in the ribosome and presence of its peptide control the binding and GTPase activity of these translation factors. The results explain how idling GTPase activity and negative interference between different translation factors are avoided and suggest that hybrid sites for tRNA on the ribosome play essential roles in translocation of tRNAs, recycling of class 1 release factors by RF3, and recycling of ribosomes back to a new round of initiation. We also propose a model for translocation of tRNAs in two separate steps, which clarifies the roles of EF-G·GTP and GTP hydrolysis in this process.

We have studied the kinetics of transcription through a nucleosome core. RNA polymerase transcribes the first approximately 25 bp of nucleosomal DNA rapidly, but then hits a barrier and continues slowly to the nucleosomal dyad region. Here, the barrier disappears and the transcript is completed at a rapid rate, as if on free DNA, indicating that histone octamer transfer is completed as polymerase reaches the dyad. If DNA behind the polymerase is removed during transcription, the barrier does not appear until the polymerase has penetrated up to 15 bp farther into the nucleosome. On a longer template, the barrier is almost eliminated. We have shown previously that the octamer is transferred around the transcribing polymerase via an intermediate containing an intranucleosomal DNA loop. Our results exclude the possibility that polymerase has difficulty breaking histone-DNA contacts and suggest instead that polymerase pauses because it has difficulty transcribing DNA in the loop.

Department of Cell Biology New York University School of Medicine New York, New York 10016 The purpose of this review is to assess present knowledge about the mechanism by which eucar- yotic ribosomes select the correct site for initiation of protein synthesis. Although the available evi- dence is insufficient to define the mechanism une- quivocally, certain features of translational initia- tion in eucaryotes are sufficiently compelling to warrant discussion. I have attempted to formulate a working model which, although speculative, ac- counts for most of the peculiarities of eucaryotic initiation. It seems particularly worth emphasizing that eucaryotic translational systems have unique characteristics not observed in procaryotes, and therefore eucaryotes might use novel mechanisms for initiation.

The 3′→5′ exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the ϕ29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic α-like DNA polymerases, specifically inactivated the 3′→5′ exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed., T h i s i n v e s t i g a t i o n h a s b e e n a i d e d b y r e s e a r c h g r a n t 5 R O l G M 2 7 2 4 2 - 0 9 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , b y g r a n t n u m b e r P B 8 7 0 3 2 3 f r o m D i r e c c i 6 n G e n e r a l d e l n v e s t i g a c i d n C i e n t i f i c a y T h c n i c a , a n d b y g r a n t s f r o m F o n d o d e l n v e s t i g a c i o n e s S a n i t a r i a s a n d F u n d a c i 6 n R a - m 6 n A r e c e s . A . R a n d G . M . w e r e F e l l o w s f r o m C o n s e j o S u p e r i o r d e l n v e s t i g a c i o n e s C i e n t i f i c a s - C a j a d e A h o r r o s d e M a d r i d a n d f r o m t h e S p a n i s h - F r e n c h M e r c u r e P r o g r a m , r e s p e c t i v e l y . T h e c o s t s o f p u b l i c a t i o n o f t h i s a r t i c l e w e r e d e f r a y e d i n p a r t b y t h e p a y m e n t o f p a g e c h a r g e s . T h i s a r t i c l e m u s t t h e r e f o r e b e h e r e b y m a r k e d “a d v e r f i s e m e n f” i n a c c o r d a n c e w i t h 1 8 U . S . C . S e c t i o n 1 7 3 4 s o l e l y t o i n d i c a t e t h i s f a c t .