Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in infection1 and a common target of antiretrovirals2. The reaction is catalyzed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion along with 2 copies of dimeric viral genomic RNA5 and host tRNALys3, which acts as a primer for initiation of reverse transcription6,7. Upon viral entry, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis for RT function during initiation has remained a mystery. Here we apply cryo-electron microscopy (cryo-EM) to determine a three-dimensional structure of the HIV-1 RT initiation complex. RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5′ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5 ′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNA Lys 3 . Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

The initiation of reverse transcription in human immunodeficiency virus-1 is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNA as a primer for initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription initiation kinetics. Using single-molecule Förster resonance energy transfer spectroscopy, we demonstrate that the vRNA-tRNA initiation complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid-RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA-tRNA primer binding site helix from 18 base pairs to 22 base pairs stabilizes RT binding to the complex and that the tRNA 5' end has a role in modulating RT binding. RT occupancy on the complex stabilizes helix 1 formation and reduces global structural heterogeneity. The stabilization of helix 1 upon RT binding may serve to destabilize helix 2, the first pause site for RT during initiation, during later steps of reverse transcription initiation.

cement fibrils. These materials are characterized by AFM-based nanomechanical measurements and compared to the Wild-Type barnacle adhesive. Synthetic and recombinant adhesive materials provide a route to scale up and study a scarce but potent class of multifunctional adhesive nanostructures produced by one of the most tenacious marine fouling organisms in the ocean.

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.