Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs.

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNASec , which is subsequently converted by selenocysteine synthase to Sec-tRNASec . Escherichia coli SerRS serylates tRNASec poorly; this may hinder efficient production of designer selenoproteins in vivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNASec synthesis. They display 10-, 8-, and 4-fold increased kcat /KM values compared to wild-type SerRS using synthetic tRNASec species as substrates. The enzyme variants also facilitate in vivo read-through of a UAG codon in the position of the critical serine146 of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.

Upgrading aminoacyl-tRNA synthetases for genetic code expansion.

Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the l-α-amino acid structure (e.g. d-amino acid or ß-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion.

A Facile Method for Producing Selenocysteine-Containing Proteins.

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer . Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo-tRNAUTu system offers a new selenoprotein engineering platform.

Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli.

Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNASec) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNASec requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNASec for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the ß' clamp domain plays the most prominent role. In this work, we investigate the role of the ß gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Learning from the Leaders: Gene Regulation by the Transcription Termination Factor Rho.

The RNA helicase Rho triggers 20-30% of transcription termination events in bacteria. While Rho is associated with most transcription elongation complexes, it only promotes termination of a subset. Recent studies of individual Rho-dependent terminators located within the 5' leader regions of bacterial mRNAs have identified novel mechanisms that govern Rho target specificity and have revealed unanticipated physiological functions for Rho. In particular, the multistep nature of Rho-dependent termination enables regulatory input from determinants beyond the sequence of the Rho loading site, and allows a given Rho-dependent terminator to respond to multiple signals. Further, the unique position of Rho as a sensor of cellular translation has been exploited to regulate the transcription of genes required for protein synthesis, including those specifying Mg(2+) transporters.

An RNA motif advances transcription by preventing Rho-dependent termination.

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.

When Too Much ATP Is Bad for Protein Synthesis.

Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg(2+)), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg(2+) limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg(2+) is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg(2+) limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg(2+) to indispensable cellular processes.

An α helix to ß barrel domain switch transforms the transcription factor RfaH into a translation factor.

NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a ß barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-ß conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface.

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.

The ß subunit gate loop is required for RNA polymerase modification by RfaH and NusG.

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the ß gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the ß' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.

Functional regions of the N-terminal domain of the antiterminator RfaH.

RfaH is a bacterial elongation factor that increases expression of distal genes in several long, horizontally acquired operons. RfaH is recruited to the transcription complex during RNA chain elongation through specific interactions with a DNA element called ops. Following recruitment, RfaH remains bound to RNA polymerase (RNAP) and acts as an antiterminator by reducing RNAP pausing and termination at some factor-independent and Rho-dependent signals. RfaH consists of two domains connected by a flexible linker. The N-terminal RfaH domain (RfaH(N)) recognizes the ops element, binds to the RNAP and reduces pausing and termination in vitro. Functional analysis of single substitutions in this domain reported here suggests that three separate RfaH(N) regions mediate these functions. We propose that a polar patch on one side of RfaH(N) interacts with the non-template DNA strand during recruitment, whereas a hydrophobic surface on the opposite side of RfaH(N) remains bound to the beta' subunit clamp helices domain throughout transcription of the entire operon. The third region is apparently dispensable for RfaH binding to the transcription complex but is required for the antitermination modification of RNAP.