Ribosomal protein S1 unwinds double-stranded RNA in multiple steps.
ABSTRACT: The sequence and secondary structure of the 5'-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5'-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure ("stand-by site") before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.
Project description:In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30?S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (?100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.
Project description:Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.
Project description:During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5' untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA.
Project description:Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.
Project description:Viral RNA helicases of the NS3/NPH-II group unwind RNA duplexes by processive, directional translocation on one of the duplex strands. The translocation is preceded by a poorly understood unwinding initiation phase. For NPH-II from vaccinia virus, unwinding initiation is rate limiting for the overall unwinding reaction. To develop a mechanistic understanding of the unwinding initiation, we studied kinetic and thermodynamic aspects of this reaction phase for NPH-II in vitro, using biochemical and single molecule fluorescence approaches. Our data show that NPH-II functions as a monomer and that different stages of the ATP hydrolysis cycle dictate distinct binding preferences of NPH-II for duplex versus single-stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single conformation but rather at least two distinct conformations in each of the analyzed stages of ATP hydrolysis. These conformations interconvert with rate constants that depend on the stage of the ATP hydrolysis cycle. Our data establish a basic mechanistic framework for unwinding initiation by NPH-II and suggest that the various stages of the ATP hydrolysis cycle do not induce single, stage-specific conformations in the NPH-II-RNA complex but primarily control transitions between multiple states.
Project description:Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3' and 5' ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.
Project description:Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.
Project description:Pif1 is a prototypical member of the 5' to 3' DNA helicase family conserved from bacteria to human. It has a high binding affinity for DNA, but unwinds double-stranded DNA (dsDNA) with a low processivity. Efficient DNA unwinding has been observed only at high protein concentrations that favor dimerization of Pif1. In this research, we used single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) to study the DNA unwinding activity of Saccharomyces cerevisiae Pif1 (Pif1) under different forces exerted on the tails of a forked dsDNA. We found that Pif1 can unwind the forked DNA repetitively for many unwinding-rezipping cycles at zero force. However, Pif1 was found to have a very limited processivity in each cycle because it loosened its strong association with the tracking strand readily, which explains why Pif1 cannot be observed to unwind DNA efficiently in bulk assays at low protein concentrations. The force enhanced the unwinding rate and the total unwinding length of Pif1 significantly. With a force of 9 pN, the rate and length were enhanced by more than 3- and 20-fold, respectively. Our results imply that the DNA unwinding activity of Pif1 can be regulated by force. The relevance of this characteristic of Pif1 to its cellular functions is discussed.
Project description:RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.
Project description:DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.