Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection

The impact of point mutations in the human androgen receptor : classification of mutations on the basis of transcriptional activity

Peer reviewedPublisher PD

Allosteric Modulators of Steroid Hormone Receptors : Structural Dynamics and Gene Regulation

Peer reviewedPublisher PD

PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers.

BACKGROUND: Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). OBJECTIVES: After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. METHODS: LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na(+))-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. RESULTS: Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c(+/-)). Resting membrane potential was more depolarized in Pitx2c(+/-) atria, and TWIK-related acid-sensitive K(+) channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide's effects between wild-type and Pitx2c(+/-) atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. CONCLUSIONS: PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation

Origins of robustness in translational control via eukaryotic translation initiation factor (eIF) 2

Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. This pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness-conferring features. Our analyses indicate that robustness is a distributed property, rather than arising from the properties of any one individual pathway species. However, robustness-conferring properties are unevenly distributed between the different species, and we identify a guanine nucleotide dissociation inhibitor (GDI) complex as a species that likely contributes strongly to the robustness of the pathway. Our analyses make further predictions on the dynamic response to different types of kinases that impinge on eIF2

Origins of robustness in translational control via eukaryotic translation initiation factor (eIF) 2

Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. This pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness-conferring features. Our analyses indicate that robustness is a distributed property, rather than arising from the properties of any one individual pathway species. However, robustness-conferring properties are unevenly distributed between the different species, and we identify a guanine nucleotide dissociation inhibitor (GDI) complex as a species that likely contributes strongly to the robustness of the pathway. Our analyses make further predictions on the dynamic response to different types of kinases that impinge on eIF2

Mutational analysis of the human androgen receptor transactivation domain

Mutants were created within the main activation function domain to investigate the structure and function of this region. Structural studies based on limited proteolysis and fluorescence spectroscopy experiments, indicate that though the N-terminus is not as specially structured as either the LBD or DBD, there are regions of specific folding within the activation domain.  Results from in silco investigation suggest several possible regions of a helix within the AF-1 domain, and mutants designed to disrupt these regions were less folded than the wild-type protein. Protein-protein interaction studies showed that the four mutants designed to potentially disrupt function rather than structure, had reduced binding to the large subunit of TFIIF - RAP74, but had no effect on binding to the transcriptional co-activator SRC-1a. In a functional assay performed in yeast cells, these four same mutants all showed reduced activity, showing the same trend as binding to RAP74. This could be an indication that the function of the AR is dependent upon binding to the general transcription factor TFIIF. Interestingly one of the mutants that was found to show increased structure over the wild-type protein was previously shown to have a reduced interaction with RAP74. This implies that structure is important for interaction with the transcription machinery. This was confirmed by FTIR experiments which can detect changes in the proportion of secondary structure present in a protein. These data show that the proportion of a helix present in the AF-1 domain increases when it is complexed with RAP74. GST pull-down assays then demonstrated that the complex of AF-1 and RAP74 enhanced the binding of the co-activator SRC-1a. This is the first time that this cooperativity has been demonstrated with nuclear receptors and interacting proteins. Additionally, specific phosphorylation of the AF-1 domain by glycogen synthase kinase 3 also increases the level of binding with SRC-1a. These data together suggest a possible mechanism of action for the androgen receptor and its involvement in regulating transcription.EThOS - Electronic Theses Online ServiceGBUnited Kingdo