Major-groove sequence-specific RNA recognition by LoaP, a paralog of transcription elongation factor NusG.

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding ß-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.

Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.

Transcription-coupled global genomic repair in E. coli.

The nucleotide excision repair (NER) pathway removes helix-distorting lesions from DNA in all organisms. Escherichia coli has long been a model for understanding NER, which is traditionally divided into major and minor subpathways known as global genome repair (GGR) and transcription-coupled repair (TCR), respectively. TCR has been assumed to be mediated exclusively by Mfd, a DNA translocase of minimal NER phenotype. This review summarizes the evidence that shaped the traditional view of NER in bacteria, and reviews data supporting a new model in which GGR and TCR are inseparable. In this new model, RNA polymerase serves both as the essential primary sensor of bulky DNA lesions genome-wide and as the delivery platform for the assembly of functional NER complexes in living cells.

Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

Introduction: The rpoCY75N mutation in the zinc-binding domain of the ß' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022. Materials and Methods: Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector. Results: Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage. Conclusions: The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.

In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Roles of zinc-binding domain of bacterial RNA polymerase in transcription.

Transcription is an essential and multistep process carried out by RNA polymerase (RNAP). In bacterial RNAP, in addition to the catalytic core domain, multiple other conserved domains are also identified to play regulatory roles in transcription. One such domain is the zinc-binding domain (ZBD) located at the N terminus of the largest subunit ß' in bacterial RNAP, whose homolog is also reported in eukaryotic RNAPs. Recent structural and biochemical studies have revealed various key roles of the conserved ß' ZBD during different steps of transcription. In this review, we summarize recent progress on the regulatory roles of this ß' ZBD in bacterial transcription.

Conjugation Operons in Gram-Positive Bacteria with and without Antitermination Systems.

Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators inside the conjugation operon to be bypassed, but also the differential expression of a subset of genes. Here, we show that some conjugation operons on plasmids belonging to the Inc18 family of Gram-positive broad host-range plasmids do not possess an antitermination system, suggesting that the absence of an antitermination system may have advantages. The possible (dis)advantages of conjugation operons possessing (or not) an antitermination system are discussed.

A NusG Specialized Paralog That Exhibits Specific, High-Affinity RNA-Binding Activity.

Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, while the C-terminal domain (CTD) forms dynamic interactions with Rho, S10, NusB and NusA to affect transcription elongation. While virtually all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Herein, we discover that LoaP requires a small RNA hairpin located within the 5' leader region of its targeted operons. LoaP binds the RNA element with nanomolar affinity and high specificity, in contrast to other NusG proteins, which have not been shown to exhibit RNA-binding activity. These data reveal a sequence feature that can be used to identify LoaP-regulated operons. This discovery also expands the repertoire of macromolecular interactions exhibited by the NusG CTD during transcription elongation to include an RNA ligand.

Resolving the activation mechanism of the D99N antiterminator LicT protein.

LicT is an antiterminator protein of the BglG family whose members are key players in the control of carbohydrate catabolism in bacteria. These antiterminators are generally composed of three modules, an N-terminal RNA-binding domain (CAT) followed by two homologous regulation modules (PRD1 and PRD2) that control the RNA binding activity of the effector domain via phosphorylation on conserved histidines. Although several structures of isolated domains of BglG-like proteins have been described, no structure containing CAT and at least one PRD simultaneously has yet been reported in an active state, precluding detailed understanding of signal transduction between modules. To fulfill this gap, we recently reported the complete NMR sequence assignment of a constitutively active mutant (D99N) CAT-PRD1*, which contains the effector domain and the first regulation domain of LicT. As a follow-up, we have determined and report here the 3D solution structure of this active, dimeric LicT construct (40 kDa). The structure reveals how the mutation constrains the PRD1 regulation domain into an active conformation which is transduced to CAT via a network of negatively charged residues belonging to PRD1 dimeric interface and to the linker region. In addition, our data support a model where BglG-type antitermination regulatory modules can only adopt a single conformation compatible with the active structure of the effector domain, regardless of whether activation is mediated by mutation on the first or second PRD. The linker between the effector and regulation modules appears to function as an adaptable hinge tuning the position of the functional modules.

Origins and Molecular Evolution of the NusG Paralog RfaH.

The only universally conserved family of transcription factors comprises housekeeping regulators and their specialized paralogs, represented by well-studied NusG and RfaH. Despite their ubiquity, little information is available on the evolutionary origins, functions, and gene targets of the NusG family members. We built a hidden Markov model profile of RfaH and identified its homologs in sequenced genomes. While NusG is widespread among bacterial phyla and coresides with genes encoding RNA polymerase and ribosome in all except extremely reduced genomes, RfaH is mostly limited to Proteobacteria and lacks common gene neighbors. RfaH activates only a few xenogeneic operons that are otherwise silenced by NusG and Rho. Phylogenetic reconstructions reveal extensive duplications and horizontal transfer of rfaH genes, including those borne by plasmids, and the molecular evolution pathway of RfaH, from "early" exclusion of the Rho terminator and tightened RNA polymerase binding to "late" interactions with the ops DNA element and autoinhibition, which together define the RfaH regulon. Remarkably, NusG is not only ubiquitous in Bacteria but also common in plants, where it likely modulates the transcription of plastid genes.IMPORTANCE In all domains of life, NusG-like proteins make contacts similar to those of RNA polymerase and promote pause-free transcription yet may play different roles, defined by their divergent interactions with nucleic acids and accessory proteins, in the same cell. This duality is illustrated by Escherichia coli NusG and RfaH, which silence and activate xenogenes, respectively. We combined sequence analysis and recent functional and structural insights to envision the evolutionary transformation of NusG, a core regulator that we show is present in all cells using bacterial RNA polymerase, into a virulence factor, RfaH. Our results suggest a stepwise conversion of a NusG duplicate copy into a sequence-specific regulator which excludes NusG from its targets but does not compromise the regulation of housekeeping genes. We find that gene duplication and lateral transfer give rise to a surprising diversity within the only ubiquitous family of transcription factors.

Transcription termination and antitermination of bacterial CRISPR arrays.

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences ('repeats') interspersed with short, variable sequences ('spacers'). CRISPR arrays are transcribed and processed into individual CRISPR RNAs that each include a single spacer, and direct Cas proteins to complementary sequences in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho can prematurely terminate transcription of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.

Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis.

Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.

Natural pyrrolysine-biased translation of stop codons in mitochondrial peptides entirely coded by expanded codons.

During the noncanonical deletion transcription, k nucleotides are systematically skipped/deleted after each transcribed trinucleotide producing deletion-RNAs (delRNAs). Peptides matching delRNAs either result from (a) canonical translation of delRNAs; or (b) noncanonical translation of regular transcripts along expanded codons. Only along frame "0" (start site) (a) and (b) produce identical peptides. Here, mitochondrial mass spectrometry data analyses assume expanded codon/del-transcription with 3 + k (k from 0 to 12) nucleotides. Detected peptides map preferentially on previously identified delRNAs. More peptides were detected for k (1-12) when del-transcriptional and expanded codon translations start sites coincide (i.e. the 0th frame) than for frames +1 or +2. Hence, both (a) and (b) produced peptides identified here. Biases for frame 0 decrease for k > 2, reflecting codon/anticodon expansion limits. Further analyses find preferential pyrrolysine insertion at stop codons, suggesting Pyl-specific mitochondrial suppressor tRNAs loaded by Pyl-specific tRNA synthetases with unknown origins. Pyl biases at stops are stronger for regular than expanded codons suggesting that Pyl-tRNAs are less competitive with near-cognate tRNAs in expanded codon contexts. Statistical biases for these findings exclude that detected peptides are experimental and/or bioinformatic artefacts implying both del-transcription and expanded codons translation occur in human mitochondria.

A Tale of Good Fortune in the Era of DNA.

Two strains of good fortune in my career were to stumble upon the Watson-Gilbert laboratory at Harvard when I entered graduate school in 1964, and to study gene regulation in bacteriophage λ when I was there. λ was almost entirely a genetic item a few years before, awaiting biochemical incarnation. Throughout my career I was a relentless consumer of the work of previous and current generations of λ geneticists. Empowered by this background, my laboratory made contributions in two areas. The first was regulation of early gene transcription in λ, the study of which began with the discovery of the Rho transcription termination factor, and the regulatory mechanism of transcription antitermination by the λ N protein, subjects of my thesis work. This was developed into a decades-long program during my career at Cornell, studying the mechanism of transcription termination and antitermination. The second area was the classic problem of prophage induction in response to cellular DNA damage, the study of which illuminated basic cellular processes to survive DNA damage.

Universal Actuator for Efficient Silencing of Escherichia coli Genes Based on Convergent Transcription Resistant to Rho-Dependent Termination.

Dynamic control is a distinguished strategy in modern metabolic engineering, in which inducible convergent transcription is an attractive approach for conditional gene silencing. Instead of a simple strong "reverse" (r-) promoter, a three-component actuator has been developed for constitutive genes silencing. These actuators, consisting of r-promoters with different strengths, the ribosomal transcription antitermination-inducing sequence rrnG-AT, and the RNase III processing site, were inserted into the 3'-UTR of three E. coli metabolic genes. Second and third actuator components were important to improve the effectiveness and robustness of the approach. The maximal silencing folds achieved for gltA, pgi, and ppc were approximately 7, 11, and >100, respectively. Data were analyzed using a simple model that considered RNA polymerase (RNAP) head-on collisions as the unique reason for gene silencing and continued transcription after collision with only one of two molecules. It was previously established that forward (f-) RNAP with a trailing ribosome was approximately 13-times more likely to continue transcription after head-on collision than untrailed r-RNAP which is sensitive to Rho-dependent transcription termination (RhoTT). According to the current results, this bias in complex stabilities decreased to no more than (3.0-5.7)-fold if r-RNAP became resistant to RhoTT. Therefore, the developed constitutive actuator could be considered as an improved tool for controlled gene expression mainly due to the transfer of r-transcription into a state that is resistant to potential termination and used as the basis for the design of tightly regulated actuators for the achievement of conditional silencing.

NMR chemical shift assignment of a constitutively active fragment of the antitermination protein LicT.

LicT belongs to an essential family of bacterial antitermination proteins which bind to nascent mRNAs in order to stimulate transcription of sugar-metabolizing operons. As most of other antitermination proteins involved in carbohydrate metabolism, LicT is composed of a N-terminal RNA-binding module (CAT) and two homologous regulatory modules (PRD1 and PRD2). The activity of the CAT effector module is controlled by antagonist phosphorylations by the phosphotransferase system on conserved histidines of the two C-terminal PRDs in response to available carbon sources. Previous studies on truncated and mutant constructs have provided partial structural insight into the mechanism of signal transduction between the N-terminal RNA-binding domain and the two regulation modules. However, no structure at atomic resolution has been ever solved that contain the RNA-binding domain and a regulation module. We report the NMR assignment of a constitutively active fragment of LicT, named D99A-CAT-PRD1 or CAT-PRD1*. This fragment is composed of the RNA-binding module and the first N-terminal regulation module which bears the mutation of Asp99 to an asparagine. It is dimeric as the native protein, with a 40 kD molecular weight. The D99N mutation is sufficient to endow this fragment with a high RNA-binding constitutive activity, in a phosphorylation-free context. The assignment reported here should set the base of future NMR investigation of signal transduction between the regulatory module and the effector module in the active state of the protein, and in the long term enable the structural study of the full length protein structure in interaction with its target RNA.

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation.

LicT belongs to an essential family of bacterial transcriptional antitermination proteins controlling the expression of sugar-metabolizing operons. When activated, they bind to nascent mRNAs, preventing premature arrest of transcription. The RNA binding capacity of the N-terminal domain CAT is controlled by phosphorylations of two homologous regulation modules by the phosphotransferase system (PTS). Previous studies on truncated and mutant proteins provided partial insight into the mechanism of signal transduction between the effector and regulatory modules. We report here the conformational and functional investigation on the allosteric activation of full-length LicT. Combining fluorescence anisotropy and NMR, we find a tight correlation between LicT RNA binding capacity and CAT closure upon PTS-mediated phosphorylation and phosphomimetic mutations. Our study highlights fine structural differences between activation processes. Furthermore, the NMR study of full-length proteins points to the back and forth propagation of structural restraints from the RNA binding to the distal regulatory module.

Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase.

Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to co-opt NusA in doing so.

NusG, an Ancient Yet Rapidly Evolving Transcription Factor.

Timely and accurate RNA synthesis depends on accessory proteins that instruct RNA polymerase (RNAP) where and when to start and stop transcription. Among thousands of transcription factors, NusG/Spt5 stand out as the only universally conserved family of regulators. These proteins interact with RNAP to promote uninterrupted RNA synthesis and with diverse cellular partners to couple transcription to RNA processing, modification or translation, or to trigger premature termination of aberrant transcription. NusG homologs are present in all cells that utilize bacterial-type RNAP, from endosymbionts to plants, underscoring their ancient and essential function. Yet, in stark contrast to other core RNAP components, NusG family is actively evolving: horizontal gene transfer and sub-functionalization drive emergence of NusG paralogs, such as bacterial LoaP, RfaH, and UpxY. These specialized regulators activate a few (or just one) operons required for expression of antibiotics, capsules, secretion systems, toxins, and other niche-specific macromolecules. Despite their common origin and binding site on the RNAP, NusG homologs differ in their target selection, interacting partners and effects on RNA synthesis. Even among housekeeping NusGs from diverse bacteria, some factors promote pause-free transcription while others slow the RNAP down. Here, we discuss structure, function, and evolution of NusG proteins, focusing on unique mechanisms that determine their effects on gene expression and enable bacterial adaptation to diverse ecological niches.

Vibrio cholerae YaeO is a Structural Homologue of RNA Chaperone Hfq that Inhibits Rho-dependent Transcription Termination by Dissociating its Hexameric State.

Rho-dependent transcription termination is a well-conserved process in bacteria. The Psu and YaeO proteins are the two established inhibitors of the ATP-dependent RNA helicase Rho protein of Escherichia coli. Here, we show a detailed sequence and phylogenetic analysis demonstrating that Vibrio cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO induces significant growth defect on in vivo expression and inhibits in vitro functions of the V. cholerae Rho on directly binding to the latter. Through various biophysical techniques, we showed that interaction of VcYaeO disrupts the oligomeric state of the VcRho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR structure of the EcYaeO. Interestingly, VcYaeO structurally resembles the Hfq protein, and like the latter, it exhibits ssDNA/RNA-binding properties. Docking studies demonstrate probable interactions of VcYaeO with VcRho and mode of inhibition of RNA binding to Rho. We propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter's hexameric assembly and also likely by sequestering the RNA from the Rho primarybinding sites.