Carlos Alvarado

All Publications

• eIF1 and eIF5 dynamically control translation start site fidelity. Nature structural & molecular biology Grosely, R., Alvarado, C., Ivanov, I. P., Nicholson, O. B., Puglisi, J. D., Dever, T. E., Lapointe, C. P. 2025

  Abstract

  Human translation initiation requires accurate recognition of translation start sites. While AUG codons are canonical start sites, non-AUG codons are also used, typically with lower efficiency. The initiator tRNA and initiation factors eIF1 and eIF5 control recognition. How they distinguish different start sites yet allow flexible recognition remains unclear. Here we used real-time single-molecule assays and an in vitro reconstituted human system to reveal how eIF1 and eIF5 direct start site selection. eIF1 binds initiation complexes in two modes: stable binding during scanning, followed by transient, concentration-dependent rebinding after start site recognition. Termination of eIF1 rebinding requires transient and concentration-dependent binding by eIF5, which allows the formation of translation competent ribosomes. Non-AUG start sites differentially stabilize eIF1 and destabilize eIF5 binding, blocking initiation at multiple points. We confirmed these opposing effects in human cells. Collectively, our findings uncover that eIF1 and eIF5 directly compete to bind initiation complexes and illuminate how their dynamic interplay tunes the fidelity of start site recognition, which has broad connections to health and disease.

  View details for DOI 10.1038/s41594-025-01629-y

  View details for PubMedID 40721534

  View details for PubMedCentralID 363659

• Conserved long-range interactions are required for stable folding of orthoflaviviral genomic RNA. Nucleic acids research Palo, M. Z., Ha, B., Lapointe, C. P., Alvarado, C., Janetzko, J., Carette, J. E., Puglisi, J. D., Puglisi, E. V. 2025; 53 (11)

  Abstract

  Long-range tertiary interactions are a widespread structural feature in viral RNAs (vRNAs) and mRNAs. In the orthoflaviviruses, conserved complementary sequences in the 5' and 3' terminal regions have an essential role in viral replication. Long-range pairing of these conserved sequences is proposed to facilitate a switch between two alternative vRNA conformations. Yet the detailed nature of these interactions, their relative populations and their exchange are required to formulate a mechanistic model of their role in regulation of the viral life cycle. Here, we used single-molecule Förster resonance energy transfer to study the global conformation of vRNAs by measuring their end-to-end distances. We observed that vRNA conformation is heterogeneous, and that conformers with close end-to-end distances have unusual kinetic stability when compared with mRNA lacking these specific long-range interactions. vRNAs also partition between at least two stable states with a large rearrangement of the terminal regions (>50 Å change in end-to-end distance). We demonstrate that this bistability depends on long-range interactions and is modulated by host factors such as the initiation factor complex eIF4F. Understanding how vRNA and its stability is influenced by interactions with other host and viral factors will help to elucidate a mechanistic role for these highly conserved orthoflaviviral sequences.

  View details for DOI 10.1093/nar/gkaf514

  View details for PubMedID 40521663

  View details for PubMedCentralID PMC12168079

• Structure-based discovery of hydrocarbon-stapled paxillin peptides that block FAK scaffolding in cancer. Nature communications Reyes, L., Naser, L., Weiner, W. S., Thifault, D., Stahl, E., McCreary, L., Nott, R., Quick, C., Buchberger, A., Alvarado, C., Rivera, A., Miller, J. A., Khatiwala, R., Cherry, B. R., Nelson, R., Martin-Garcia, J. M., Stephanopoulos, N., Fromme, R., Fromme, P., Cance, W., Marlowe, T. 2025; 16 (1): 2060

  Abstract

  The focal adhesion kinase (FAK) scaffold provides FAK-targeted cancer therapeutics with greater efficacy and specificity than traditional kinase inhibitors. The FAK scaffold function largely involves the interaction between FAK's focal adhesion targeting (FAT) domain and paxillin, ultimately regulating many hallmarks of cancer. We report the design of paxillin LD-motif mimetics that successfully inhibit the FAT-paxillin interaction. Chemical and biochemical screening identifies stapled peptide 1907, a high affinity binder of the FAT four-helix bundle with ~100-fold greater binding affinity than the native LD2-sequence. The X-ray co-crystal structure of the FAT-1907 complex is solved. Myristoylated 1907-analog, peptide 2012, delocalizes FAK from focal adhesions, induces cancer cell apoptosis, reduces in vitro viability and invasion, and decreases tumor burden in B16F10 melanoma female mice. Enzymatic FAK inhibition produces no comparable effects. Herein, we describe a biologically potent therapeutic strategy to target the FAK-paxillin complex, a previously deemed undruggable protein-protein interaction.

  View details for DOI 10.1038/s41467-025-57196-9

  View details for PubMedID 40021642

  View details for PubMedCentralID PMC11871066

• eIF1 and eIF5 dynamically control translation start site fidelity. bioRxiv : the preprint server for biology Grosely, R., Alvarado, C., Ivanov, I. P., Nicholson, O. B., Puglisi, J. D., Dever, T. E., Lapointe, C. P. 2024

  Abstract

  Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNAi Met on the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid (~ 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5' untranslated region (5'UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.

  View details for DOI 10.1101/2024.07.10.602410

  View details for PubMedID 39026837

  View details for PubMedCentralID PMC11257575

• Long-range RNA interactions in flavivirus replication Palo, M. Z., Ha, B., Lapointe, C., Alvarado, C., Puglisi, J. D., Puglisi, E. CELL PRESS. 2024: 85A

  View details for Web of Science ID 001194120700410

• Impacts of HIV-1 5′ UTR structure on mRNA translation initiation Stackhouse, C. I., Jackson, L. N., Alvarado, C., Grosely, R., Puglisi, J. D., Puglisi, E. CELL PRESS. 2024: 360A-361A

  View details for Web of Science ID 001194120702165

• Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation. Cell Wang, J., Shin, B., Alvarado, C., Kim, J., Bohlen, J., Dever, T. E., Puglisi, J. D. 2022

  Abstract

  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. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at 100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward 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.

  View details for DOI 10.1016/j.cell.2022.10.005

  View details for PubMedID 36334590

• eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining. Nature Lapointe, C. P., Grosely, R., Sokabe, M., Alvarado, C., Wang, J., Montabana, E., Villa, N., Shin, B., Dever, T. E., Fraser, C. S., Fernandez, I. S., Puglisi, J. D. 2022

  Abstract

  Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. 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 theuniversally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-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 translation initiation in humans.

  View details for DOI 10.1038/s41586-022-04858-z

  View details for PubMedID 35732735