364 research outputs found
Non-canonical Binding Site for Bacterial Initiation Factor 3 on the Large Ribosomal Subunit
Get PDFCanonical translation initiation in bacteria entails the assembly of the 30S initiation complex (IC), which binds the 50S subunit to form a 70S IC. IF3, a key initiation factor, is recruited to the 30S subunit at an early stage and is displaced from its primary binding site upon subunit joining. We employed four different FRET pairs to monitor IF3 relocation after 50S joining. IF3 moves away from the 30S subunit, IF1 and IF2, but can remain bound to the mature 70S IC. The secondary binding site is located on the 50S subunit in the vicinity of ribosomal protein L33. The interaction between IF3 and the 50S subunit is largely electrostatic with very high rates of IF3 binding and dissociation. The existence of the non-canonical binding site may help explain how IF3 participates in alternative initiation modes performed directly by the 70S ribosomes, such as initiation on leaderless mRNAs or re-initiation
Directional transition from initiation to elongation in bacterial translation
Get PDFThe transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selec-tion during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a tool-box of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit join-ing, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNAfMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNAfMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initia-tion
Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance
Get PDFAbstract The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a ‘seed’ region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA
Structure of the 30S translation initiation complex coupled to paused RNA polymerase and its potential for riboregulation
No full textAbstract In many bacterial species, transcription and translation can be coupled physically, with potential impact on the rates and efficiency of gene expression. Here, we present structural evidence from cryo-EM demonstrating that a bacterial RNA polymerase that is paused proximally to the promoter can associate with the pioneering 30S translation initiation complex (30S IC). These findings suggest that the physical link between transcription and translation can be established prior to commitment to protein synthesis. Although the mRNA is embedded in this ‘early expressome’ complex, it can nonetheless interact with small regulatory RNA (sRNA) and be targeted for cleavage in the protein-coding region by the RNA degradosome assembly in vitro. The potential tagging of transcripts with sRNA during pioneering and subsequent stages of translation initiation, when the 30S IC is at the 5′ end of a polyribosome, may in principle contribute to efficient and rapid termination of gene expression in response to regulatory signals
Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2.
Get PDFGTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNAPhe, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions
Dynamics of Network Formation Processes in the Co-Author Model
Get PDFThis article studies the dynamics in the formation processes of a mutual consent network in game theory setting: the Co-Author Model. In this article, a limited observation is applied and analytical results are derived. Then, 2 parameters are varied: the number of individuals in the network and the initial probability of the links in the network in its initial state. A simulation result shows a finding that is consistent with an analytical result for a state of equilibrium while it also shows different possible equilibria.Dynamics, Network, Game Theory, Model,Simulation, Equilibrium, Complexity
Isolated propeller aeroacoustics at positive and negative thrust Author links open overlay panel
Get PDFUsing propellers in negative thrust conditions can potentially result in many benefits, such as a steeper descent, a reduced landing run, reduced community noise, energy regeneration, etc. However, the aerodynamics and aeroacoustics of propellers in this regime are not well understood. This paper presents an aeroacoustic analysis of an isolated propeller operating in both positive and negative thrust conditions, using scale-resolved lattice-Boltzmann very large eddy simulations and the Ffowcs Williams & Hawkings analogy. The propeller was operated at a constant tip Mach number so that any differences in tonal noise between positive and negative thrust conditions were due to changes in blade loading. Results showed that the flow separation around the blades in the negative thrust case led to a 2 to 6 times higher standard deviation in integrated thrust compared to the positive thrust case. The blade loading in the negative thrust case shows the amplitude of fluctuations up to 18% for inboard sections and up to 30% near the blade tip compared to the time-averaged loads. The noise in the propeller plane is 10 dB higher in the positive thrust regime than in the negative thrust regime at a given absolute thrust level of
. The lower noise at negative thrust is caused by two factors: the lower magnitude of the negative torque compared to the positive torque at a given thrust level and the shift of the blade loading inboard in the negative thrust condition due to the stall of the blade tip. Along the propeller axis, the negative thrust regime has 13-15 dB higher noise because of the increased broadband noise generated by the flow separation. In the negative thrust case, the noise along the propeller axis (89 dB) and propeller plane (92 dB) are comparable. However, this is not the case for the propulsive case. The comparison of noise in the vicinity of the propeller plane showed that using the propellers in negative thrust conditions allows for a steeper and quieter descent compared to a conventional descent; as long as the magnitude of the negative torque produced is equal to or less than the torque required to operate the propeller in a conventional landing
Inferring object states and articulation modes from egocentric videos
Get PDFWe develop algorithms for understanding objects from the point of view of interacting with them. There are two key aspects to obtaining such an understanding. First, objects can occur in different states and we need features that are sensitive to such states. Second, different objects can be articulated in different ways and we need to understand how to correctly infer their modes of articulation. We propose self and weakly supervised techniques to obtain such an understanding of objects purely through observation of how humans interact with the world around them through their hands. Our experiments on the challenging EPIC- KITCHENS dataset show the merits of using human hands as a probe for understanding objects.Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2023-05-01The student, Rishabh Goyal, accepted the attached license on 2021-04-27 at 12:26.The student, Rishabh Goyal, submitted this Thesis for approval on 2021-04-27 at 13:39.This Thesis was approved for publication on 2021-04-28 at 09:43.DSpace SAF Submission Ingestion Package generated from Vireo submission #16585 on 2021-09-16 at 17:06:08Made available in DSpace on 2021-09-17T02:34:49Z (GMT). No. of bitstreams: 2
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Aerodynamics and Far-field Noise Emissions of a Propeller in Positive and Negative Thrust Regimes at Non-zero Angles of Attack
Get PDFThis paper studies the effect of operation at non-zero angles of attack on the aerodynamic performance and far-field noise emissions of an isolated propeller operating at positive and negative thrust conditions. To achieve this, scale-resolved lattice-Boltzmann very large eddy simulations coupled with the Ffowcs Williams & Hawkings analogy have been used. The results show that when the propeller operates with a 10◦ angle of attack at the positive thrust condition, the blade loading increases on the advancing side and decreases on the retreating side, leading to a 9.6% increase in integrated thrust (when computed along the propeller axis) and a negligible increase (0.1%) in propeller efficiency. Conversely, at the negative thrust condition, the operation at 10 deg angle of attack results in a 7.9% decrease in thrust magnitude and an 11.1% reduction in energy-harvesting efficiency. In this condition, the positively cambered blade sections exhibit dynamic stall at the 10◦ angle of attack, resulting in broadband fluctuations of up to 10% of the mean loading. As a result of the opposite change in absolute blade loading in the negative thrust condition compared to the positive thrust condition at the 10◦ angle of attack, the change in the noise directivity is also the opposite. Whereas in the positive thrust case, the noise increases in the region from which the propeller is tilted away (i.e., below the propeller at a positive angle of attack), in the negative thrust case, it is the other way around. This study highlights the need to account for non-zero angles of attack in propeller design and optimization analyses
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