Kinetics of codon recognition on the ribosome by tRNAs and release factors

Ribosomes translate messenger ribonucleic acid (mRNA) into proteins in all domains of life. During the elongation phase of protein synthesis, tRNAs bind to the ribosome in a codon dependent fashion as ternary complexes consisting of a protein elongation factor (EF-Tu), guanosine triphosphate (GTP), and an aminoacylated transfer ribonucleic acid (tRNA). The first step in the termination of protein synthesis is the recognition of stop codons by release factor 1 or 2 (RF1 or 2) in order to hydrolyze the completely synthesized protein from the tRNA bound in the peptidyl (P) site of the ribosome. We have developed a fluorescence based method designed to monitor codon recognition by tRNAs and RFs in the aminoacyl (A) site of the 30S subunit of the ribosome. Using the change in fluorescence of a pyrene molecule attached to the 3' end of a short mRNA as a probe, we have investigated the kinetic mechanism of ternary complex and release factor binding to the A-site of the ribosome. Codon recognition by ternary complexes occurs as part of the second order association step between the ribosome and ternary complex. By interacting with the codon during the first encounter, competition between cognate and near or non-cognate ternary complexes is reduced and rapid screening of ternary complexes may occur. We have found that physiological concentrations of the polyamines spermine and spermidine stimulate ternary complex binding to the A- site of the ribosome at least as well as unphysiologically high concentrations of magnesium ions commonly used during in vitro translation experiments. We have also investigated the thermodynamics and kinetics of RF1 binding to the ribosome when a stop codon or a variety of sense codons were positioned in the A-site. The relative affinity of RF1 to different sense codons and the catalysis of peptide release by the RF were not directly related. The observed disparity between binding and catalysis indicates that RF1 employs an induced fit mechanism in the discrimination of stop codons from sense codon

Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA

We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10(8) M(-1)⋅s(-1). The NF-κB-DNA complex dissociates with a rate constant of 0.41 s(-1), yielding a KD of 2.8 nM. When IκBα is added to the NF-κB-DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB-DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB-DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB-DNA complex

Small Molecules CK-666 and CK-869 Inhibit Actin-Related Protein 2/3 Complex by Blocking an Activating Conformational Change

SummaryActin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo

Calorie Restriction-Induced Increase in Skeletal Muscle Insulin Sensitivity Is Not Prevented by Overexpression of the p55α Subunit of Phosphoinositide 3-Kinase

Introduction: The Phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in skeletal muscle insulin-stimulated glucose uptake. While whole-body and tissue specific knockout (KO) of individual or combinations of the regulatory subunits of PI3K (p85α, p55α, and p50α or p85β); increase insulin sensitivity, no study has examined whether increasing the expression of the individual regulatory subunits would inhibit insulin action in vivo. Therefore, the objective of this study was to determine whether skeletal muscle-specific overexpression of the p55α regulatory subunit of PI3K impairs skeletal muscle insulin sensitivity, or prevents its enhancement by caloric restriction.Methods: We developed a novel “floxed” mouse that, through the Cre-LoxP approach, allows for tamoxifen (TMX)-inducible and skeletal muscle-specific overexpression of the p55α subunit of PI3K (referred to as, ‘p55α-mOX’). Beginning at 10 weeks of age, p55α-mOX mice and their floxed littermates (referred to as wildtype [WT]) either continued with free access to food (ad libitum; AL), or were switched to a calorie restricted diet (CR; 60% of AL intake) for 20 days. We measured body composition, whole-body energy expenditure, oral glucose tolerance and ex vivo skeletal muscle insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake method.Results: p55α mRNA and protein expression was increased ∼2 fold in muscle from p55α-mOX versus WT mice. There were no differences in energy expenditure, total activity, or food intake of AL-fed mice between genotypes. Body weight, fat and lean mass, tissue weights, and fasting glucose and insulin were comparable between p55α-mOX and WT mice on AL, and were decreased equally by CR. Interestingly, overexpression of p55α did not impair oral glucose tolerance or skeletal muscle insulin signaling or sensitivity, nor did it impact the ability of CR to enhance these parameters.Conclusion: Skeletal muscle-specific overexpression of p55α does not impact skeletal muscle insulin action, suggesting that p85α and/or p50α may be more important regulators of skeletal muscle insulin signaling and sensitivity