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Alvarado, C.

Publications and source records attributed to Alvarado, C..

3 recordsLinked to original sources

Survey of activation-induced genome architecture reveals a novel enhancer of Myc

The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 hours post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

genetics↗

eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

biophysics↗

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5'UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. Once engaged with the mRNA, 43S scanning occurs at >100 nucleotides per second, independent of multiple cycles of ATP-hydrolysis by RNA helicases. The scanning ribosomes can proceed through RNA secondary structures, but 5'UTR hairpin sequences near start codons drive scanning ribosomes at start codons back in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5'UTR structures and upstream near-cognate start codons. One Sentence SummaryDirect observation of scanning eukaryotic ribosomes establishes a quantitative framework of scanning and its regulation.

biophysics↗