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Abstract Background Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. Results Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH. Conclusions We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

Persiathiacin A is a novel thiopeptide antibiotic produced by Actinokineospora species UTMC 2448. It has potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis . Thiopeptides, including persiathiacin A, exhibit antibacterial activity by inhibiting protein synthesis. In this study, we characterize the mechanism of action of persiathiacin A and investigate how resistance to this antibiotic can emerge. In vitro assays revealed that persiathiacin A inhibits translation elongation, leading to ribosome stalling. Genetic analysis of resistant Bacillus subtilis mutants identified mutations primarily in the rplK gene encoding ribosomal protein L11, which is the binding site for other 26-membered macrocycle-containing thiopeptides. The resistant mutants showed growth impairment and an increased lag time, even in the absence of persiathiacin. Comparative proteomic analysis of a resistant mutant versus the parental strain revealed multiple changes, indicative of negative effects on protein synthesis. Thus, although persiathiacin-resistant mutants can arise readily by the loss of L11 function, it is likely that such mutants would be severely compromised in pathogenesis. Furthermore, bioinformatics analysis identified differences in the key amino acids within the thiopeptide-binding region of L11 in the persiathiacin producer. These probably prevent the antibiotic from associating with its target, providing a mechanism for self-resistance.

HIV infection is one of the most acute problems of our time, characterized by slow development, prolonged course, and numerous clinical manifestations. Currently, there is a large number of drugs acting on different processes of human immunodeficiency virus replication, which constitute the group of highly active antiretroviral therapy (HAART). This article shows a theoretical review of modern HAART and analyzes the prescribed treatment regimens for patients with HIV infection. The study revealed two most common combinations: nucleoside reverse transcriptase inhibitors + protease inhibitors; nucleoside + non-nucleoside reverse transcriptase inhibitors.

Transcription elongation stalls at lesions in the DNA template 1 . For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site 2 . Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs 3 , does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd 4 acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication 5 . We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome., (© 2024. The Author(s).)

The flow of genetic information from the chromosome to protein in all living organisms consists of two steps: (1) copying information coded in DNA into an mRNA intermediate via transcription by RNA polymerase, followed by (2) translation of this mRNA into a polypeptide by the ribosome. Unlike eukaryotes, where transcription and translation are separated by a nuclear envelope, in bacterial cells, these two processes occur within the same compartment. This means that a pioneering ribosome starts translation on nascent mRNA that is still being actively transcribed by RNA polymerase. This tethering via mRNA is referred to as 'coupling' of transcription and translation (CTT). CTT raises many questions regarding physical interactions and potential mutual regulation between these large (ribosome is ~2.5 MDa and RNA polymerase is 0.5 MDa) and powerful molecular machines. Accordingly, we will discuss some recently discovered structural and functional aspects of CTT., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Insertions of transposable elements (TEs) in eukaryotic genomes are usually associated with repressive chromatin, which spreads to neighbouring genomic sequences. In ovaries of Drosophila melanogaster , the Piwi-piRNA pathway plays a key role in the transcriptional silencing of TEs considered to be exerted mostly through the establishment of H3K9me3 histone marks recruiting Heterochromatin Protein 1a (HP1a). Here, using RNA-seq, we investigated the expression of TEs and the adjacent genomic regions upon Piwi and HP1a germline knockdowns sharing a similar genetic background. We found that the depletion of Piwi and HP1a led to the derepression of only partially overlapping TE sets. Several TEs were silenced predominantly by HP1a, whereas the upregulation of some other TEs was more pronounced upon Piwi knockdown and, surprisingly, was diminished upon a Piwi/HP1a double-knockdown. We revealed that HP1a loss influenced the expression of thousands of protein-coding genes mostly not adjacent to TE insertions and, in particular, downregulated a putative transcriptional factor required for TE activation. Nevertheless, our results indicate that Piwi and HP1a cooperatively exert repressive effects on the transcription of euchromatic loci flanking the insertions of some Piwi-regulated TEs. We suggest that this mechanism controls the silencing of a small set of TE-adjacent tissue-specific genes, preventing their inappropriate expression in ovaries.

Rifamycins, such as rifampicin (Rif), are potent inhibitors of bacterial RNA polymerase (RNAP) and are widely used antibiotics. Rifamycin resistance is usually associated with mutations in RNAP that preclude rifamycin binding. However, some bacteria have a type of ADP-ribosyl transferases, Arr, which ADP-ribosylate rifamycin molecules, thus inactivating their antimicrobial activity. Here, we directly show that ADP-ribosylation abolishes inhibition of transcription by rifampicin, the most widely used rifamycin antibiotic. We also show that a natural rifamycin, kanglemycin A (KglA), which has a unique sugar moiety at the ansa chain close to the Arr modification site, does not bind to Arr from Mycobacterium smegmatis and thus is not susceptible to inactivation. We, found, however, that kanglemycin A can still be ADP-ribosylated by the Arr of an emerging pathogen, Mycobacterium abscessus. Interestingly, the only part of Arr that exhibits no homology between the species is the part that sterically clashes with the sugar moiety of kanglemycin A in M. smegmatis Arr. This suggests that M. abscessus has encountered KglA or rifamycin with a similar sugar modification in the course of evolution. The results show that KglA could be an effective antimicrobial against some of the Arr-encoding bacteria.

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorylation of the RNA polymerase II C-terminal domain Y 1 S 2 P 3 T 4 S 5 P 6 S 7 consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents. We demonstrate a key role for the Ssu72/Hos2/Set3 mechanism in the suppression of cryptic antisense transcription at the 3' end of convergent genes that are most susceptible to these defects, ensuring the fidelity of gene expression within dense genomes of simple eukaryotes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2-4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve., Competing Interests: The authors have declared that no competing interests exist.

In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

RNA polymerases (RNAPs) accomplish the first step of gene expression in all living organisms. However, the sequence divergence between bacterial and human RNAPs makes the bacterial RNAP a promising target for antibiotic development. The most clinically important and extensively studied class of antibiotics known to inhibit bacterial RNAP are the rifamycins. For example, rifamycins are a vital element of the current combination therapy for treatment of tuberculosis. Here, we provide an overview of the history of the discovery of rifamycins, their mechanisms of action, the mechanisms of bacterial resistance against them, and progress in their further development.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Accuracy of transcription is essential for productive gene expression, and the past decade has brought new understanding of the mechanisms ensuring transcription fidelity. The discovery of a new catalytic domain, the Trigger Loop, revealed that RNA polymerase can actively choose the correct substrates. Also, the intrinsic proofreading activity was found to proceed via a ribozyme-like mechanism, whereby the erroneous nucleoside triphosphate (NTP) helps its own excision. Factor-assisted proofreading was shown to proceed through an exchange of active centres, a unique phenomenon among proteinaceous enzymes. Furthermore, most recent in vivo studies have revised the roles of transcription accuracy and proofreading factors, as not only required for production of errorless RNAs, but also for prevention of frequent misincorporation-induced pausing that may cause conflicts with fellow RNA polymerases and the replication machinery., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of Escherichia coli EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the β-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase.

Transcription in all living organisms is accomplished by multi-subunit RNA polymerases (msRNAPs). msRNAPs are highly conserved in evolution and invariably share a ∼400 kDa five-subunit catalytic core. Here we characterize a hypothetical ∼100 kDa single-chain protein, YonO, encoded by the SPβ prophage of Bacillus subtilis. YonO shares very distant homology with msRNAPs, but no homology with single-subunit polymerases. We show that despite homology to only a few amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive DNA-dependent RNA polymerase. We demonstrate that YonO is a bona fide RNAP of the SPβ bacteriophage that specifically transcribes its late genes, and thus represents a novel type of bacteriophage RNAPs. YonO and related proteins present in various bacteria and bacteriophages have diverged from msRNAPs before the Last Universal Common Ancestor, and, thus, may resemble the single-subunit ancestor of all msRNAPs.

The madurastatins are pentapeptide siderophores originally described as containing an unusual salicylate-capped N-terminal aziridine ring. Isolation of madurastatin C1 (1) (also designated MBJ-0034), from Actinomadura sp. DEM31376 (itself isolated from a deep sea sediment), prompted structural reevaluation of the madurastatin siderophores, in line with the recent work of Thorson and Shaaban. NMR spectroscopy in combination with partial synthesis allowed confirmation of the structure of madurastatin C1 (1) as containing an N-terminal 2-(2-hydroxyphenyl)oxazoline in place of the originally postulated aziridine, while absolute stereochemistry was determined via Harada's advanced Marfey's method. Therefore, this work further supports Thorson and Shaaban's proposed structural revision of the madurastatin class of siderophores (madurastatins A1 (2), B1 (3), C1 (1), and MBJ-0036 (4)) as N-terminal 2-(2-hydroxyphenyl)oxazolines.

The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes. Recently, deep sequencing has become increasingly popular for the study of the transcriptome due to its lower costs, higher accuracy and single nucleotide resolution. These methods have revolutionised our understanding of prokaryotic transcriptional dynamics. Here, we review the deep sequencing and data analysis techniques that are available for the study of transcription in prokaryotes, and discuss the bioinformatic considerations of these analyses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pausing by RNA polymerase is a major mechanism that regulates transcription elongation but can cause conflicts with fellow RNA polymerases and other cellular machineries. Here, we summarize our recent finding that misincorporation could be a major source of transcription pausing in vivo, and discuss the role of misincorporation-induced pausing.

The transcription error rate estimated from mistakes in end product RNAs is 10−3–10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1–3% of all ECs in wild-type cells, and 5–7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA.

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances. In the same time RNAP can be put in various types of states, such as paused, roadblocked, backtracked, etc. The experimental system thus allows studying the effects of the ribosome on different aspects of transcription elongation and the effects by RNAP on translation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulation of transcription elongation is based on response of RNA polymerase (RNAP) to various pause signals and is modulated by various accessory factors. Here we report that a 7 kDa protein p7 encoded by bacteriophage Xp10 acts as an elongation processivity factor of RNAP of host bacterium Xanthomonas oryzae, a major rice pathogen. Our data suggest that p7 stabilizes the upstream DNA duplex of the elongation complex thus disfavouring backtracking and promoting forward translocated states of the elongation complex. The p7-induced 'pushing' of RNAP and modification of RNAP contacts with the upstream edge of the transcription bubble lead to read-through of various types of pauses and termination signals and generally increase transcription processivity and elongation rate, contributing for transcription of an extremely long late genes operon of Xp10. Forward translocation was observed earlier upon the binding of unrelated bacterial elongation factor NusG, suggesting that this may be a general pathway of regulation of transcription elongation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.

Collisions between paused transcription elongation complexes and replication forks inevitably happen, which may lead to collapse of replication fork and could be detrimental to cells. Bacterial transcription factor DksA and its cofactor alarmone ppGpp were proposed to contribute to prevention of such collisions, although the mechanism of this activity remains elusive. Here we show that DksA/ppGpp do not destabilise transcription elongation complexes or inhibit their backtracking, as was proposed earlier. Instead, we show, both in vitro and in vivo, that DksA/ppGpp increase fidelity of transcription elongation by slowing down misincorporation events. As misincorporation events cause temporary pauses, contribution to fidelity suggests the mechanism by which DksA/ppGpp contribute to prevention of collisions of transcription elongation complexes with replication forks. DksA is only the second known accessory factor, after transcription factor Gre, that increases fidelity of RNA synthesis in bacteria., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA polymerase is a complex machinery, which is further embedded in interactions with other cellular components that interplay with either the transcribed DNA (DNA polymerases, topoisomerases, etc.) or the nascent RNA (RNA processing enzymes, ribosomes, etc.). In prokaryotes, coupling of transcription and translation is thought to play many regulatory roles but the mechanistic understanding of their interactions has been hindered by the lack of a defined experimental system. Here, we describe a pure transcription-coupled-to-translation system in which control of the ribosome has been achieved through its stepwise translocation towards RNA polymerase. This system can be used to study the effects of concurrent translation on RNA chain elongation and to elucidate the interface between the two macromolecular complexes.

Arimbasseri et al., in their Comment, suggest that to terminate transcription in vivo, RNA polymerase III uses a mechanism other than hairpin-dependent termination and that properties of purified polymerase may depend on preparation procedure. Evidence suggests that our preparation is indeed different from that of other methods. Our new data suggest that, apart from hairpin-dependent termination, one or more "fail-safe" termination mechanisms may exist in the cell., (Copyright © 2014, American Association for the Advancement of Science.)

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes., (Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Fic enzymes post-translationally modify proteins through AMPylation, UMPylation, phosphorylation, or phosphocholination. They have been identified across all domains of life, and they target a myriad of proteins such as eukaryotic GTPases, unstructured protein segments, and bacterial enzymes. Consequently, they play crucial roles in eukaryotic signal transduction, drug tolerance, bacterial pathogenicity, and the bacterial stress response. Structurally, they consist of an all α-helical core domain that supports and scaffolds a structurally conserved active-site loop, which catalyses the transfer of various parts of a nucleotide cofactor to proteins. Despite their diverse substrates and targets, they retain a conserved active site and reaction chemistry. This catalytic variety came to light only recently with the crystal structures of different Fic enzymes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)