Molecular basis of mRNA delivery to the bacterial ribosome.

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

Structural insights into RNA-mediated transcription regulation in bacteria.

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Transcription factors modulate RNA polymerase conformational equilibrium.

RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it.

Macromolecular assemblies supporting transcription-translation coupling.

Coordination between the molecular machineries that synthesize and decode prokaryotic mRNAs is an important layer of gene expression control known as transcription-translation coupling. While it has long been known that translation can regulate transcription and vice-versa, recent structural and biochemical work has shed light on the underlying mechanistic basis. Complexes of RNA polymerase linked to a trailing ribosome (expressomes) have been structurally characterized in a variety of states at near-atomic resolution, and also directly visualized in cells. These data are complemented by recent biochemical and biophysical analyses of transcription-translation systems and the individual components within them. Here, we review our improved understanding of the molecular basis of transcription-translation coupling. These insights are discussed in relation to our evolving understanding of the role of coupling in cells.

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World.

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea.

Seeing gene expression in cells: the future of structural biology.

Although much is known about the machinery that executes fundamental processes of gene expression in cells, much also remains to be learned about how that machinery works. A recent paper by O'Reilly et al. reports a major step forward in the direct visualization of central dogma processes at submolecular resolution inside bacterial cells frozen in a native state. The essential methodologies involved are cross-linking mass spectrometry (CLMS) and cryo-electron tomography (cryo-ET). In-cell CLMS provides in vivo protein interaction maps. Cryo-ET allows visualization of macromolecular complexes in their native environment. These methods have been integrated by O'Reilly et al. to describe a dynamic assembly in situ between a transcribing RNA polymerase (RNAP) and a translating ribosome - a complex known as the expressome - in the model bacterium Mycoplasma pneumoniae 1. With the application of improved data processing and classification capabilities, this approach has allowed unprecedented insights into the architecture of this molecular assembly line, confirming the existence of a physical link between RNAP and the ribosome and identifying the transcription factor NusA as the linking molecule, as well as making it possible to see the structural effects of drugs that inhibit either transcription or translation. The work provides a glimpse into the future of integrative structural cell biology and can serve as a roadmap for the study of other molecular machineries in their native context.

Structural basis of transcription-translation coupling and collision in bacteria.

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The lead ribosome potentially contacts RNA polymerase (RNAP) and forms a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here, we present a series of structures representing uncoupled, coupled, and collided expressome states determined by cryo-electron microscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, which stabilizes an otherwise-variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance channel to the RNAP exit channel. In this collided complex, NusG linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Structural Basis for NusA Stabilized Transcriptional Pausing.

Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.

Structural basis of transcriptional pausing in bacteria.

Transcriptional pausing by multisubunit RNA polymerases (RNAPs) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes and is a prerequisite for transcription termination. Pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. We report three crystal structures of Thermus RNAP elemental paused elongation complexes (ePECs). The structures reveal the same relaxed, open-clamp RNAP conformation in the ePEC that may arise by failure to re-establish DNA contacts during translocation. A kinked bridge-helix sterically blocks the RNAP active site, explaining how this conformation inhibits RNAP catalytic activity. Our results provide a framework for understanding how RNA hairpin formation stabilizes the paused state and how the ePEC intermediate facilitates termination.

Ribosome engineering to promote new crystal forms.

Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

The structure of the ribosome with elongation factor G trapped in the posttranslocational state.

Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome.

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3' ends of tRNA ligands. Here we present structures, at 3.6-A and 3.5-A resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer.

Insights into translational termination from the structure of RF2 bound to the ribosome.

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.

Modified uridines with C5-methylene substituents at the first position of the tRNA anticodon stabilize U.G wobble pairing during decoding.

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.

The termination of translation.

Recent results from cryoelectron microscopy, crystallography, and biochemical experiments have shed considerable light on the process by which protein synthesis is terminated when a stop codon is reached. However, a detailed understanding of the underlying mechanisms will require higher-resolution structures of the various states involved.

Crystal structure of the ribosome recycling factor bound to the ribosome.

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines.

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

Structure of the 70S ribosome complexed with mRNA and tRNA.

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.