An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation. Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue. Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E. coli ProRS. We carried out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS. Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme. Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution.

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA Pro possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA Pro by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.

Prevention behaviors represent important public health tools to limit spread of SARS-CoV-2. Adherence with recommended public health prevention behaviors among 20000 + members of a COVID-19 syndromic surveillance cohort from the mid-Atlantic and southeastern United States was assessed via electronic survey following the 2020 Thanksgiving and winter holiday (WH) seasons. Respondents were predominantly non-Hispanic Whites (90%), female (60%), and ≥ 50 years old (59%). Non-household members (NHM) were present at 47.1% of Thanksgiving gatherings and 69.3% of WH gatherings. Women were more likely than men to gather with NHM (p < 0.0001). Attending gatherings with NHM decreased with older age (Thanksgiving: 60.0% of participants aged < 30 years to 36.3% aged ≥ 70 years [p-trend < 0.0001]; WH: 81.6% of those < 30 years to 61.0% of those ≥ 70 years [p-trend < 0.0001]). Non-Hispanic Whites were more likely to gather with NHM than were Hispanics or non-Hispanic Blacks (p < 0.0001). Mask wearing, reported by 37.3% at Thanksgiving and 41.9% during the WH, was more common among older participants, non-Hispanic Blacks, and Hispanics when gatherings included NHM. In this survey, most people did not fully adhere to recommended public health safety behaviors when attending holiday gatherings. It remains unknown to what extent failure to observe these recommendations may have contributed to the COVID-19 surges observed following Thanksgiving and the winter holidays in the United States.

The COVID-19 Community Research Partnership (CCRP) is a multisite surveillance platform designed to characterize the epidemiology of the SARS-CoV-2 pandemic. This manuscript describes the CCRP study design and methodology. The CCRP includes two prospective cohorts, one with six health systems in the mid-Atlantic and southern United States, and the other with six health systems in North Carolina. With enrollment beginning April 2020, sites invited persons within their healthcare systems as well as community members to participate in daily surveillance for symptoms of COVID-like illnesses, testing and risk behaviors. Participants with electronic health records were also asked to volunteer data access. Subsets of participants, representative of the general population and including oversampling of populations of interest, were selected for repeated at home serology testing. By October 2021, 65,739 participants (62,261 adult and 3,478 pediatric) were enrolled, with 89% providing syndromic data, 74% providing EHR data, and 70% participating in one of two serology sub-studies. An average of 62% of participants completed a daily survey at least once a week, and 55% of serology kits were returned. The CCRP provides rich regional epidemiologic data and the opportunity to more fully characterize the risks and sequelae of SARS-CoV-2 infection.

SummaryAminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to their cognate tRNAs. Specific aminoacylation is dictated by a set of recognition elements that mark tRNA molecules as substrates for particular synthetases. Escherichia coli prolyl-tRNA synthetase (ProRS) has previously been shown to recognize specific bases of tRNA Pro in both the anticodon domain, which mediate initial complex formation, and in the acceptor stem, which is proximal to the site of catalysis. In this work, we unambiguously define the molecular interaction between E. coli ProRS and the acceptor stem of cognate tRNA Pro . Oxidative cross-linking studies using 2′-deoxy-8-oxo-7,8-dihydroguanosine-containing proline tRNAs identify a direct interaction between a critical arginine residue (R144) in the active site of E. coli ProRS and the G72 residue in the acceptor stem of tRNA Pro . Assays conducted with motif 2 loop variants and tRNA mutants wherein specific atomic groups of G72 were deleted, are consistent with a functionally important hydrogen bonding network between R144 and the major groove of G72. These results taken together with previous studies suggest that breaking this key contact uncouples the allosteric interaction between the anticodon domain and the aminoacylation active site, providing new insights into the communication network that governs the synthetase-tRNA interaction.

Importance: Real-world data are needed to assess incidence and factors associated with breakthrough SARS-CoV-2 infections following vaccination. Objective: Estimate incidence of breakthrough infections and assess associations with risk factors using self-reported data from a large NC population sample. Design: Prospective observational cohort study utilizing daily online survey data to capture information about COVID-19 symptoms, testing, and vaccination status. Setting: Six health care systems in North Carolina with data collected between January 15, 2021 and September 24, 2021. Participants: Adult study participants who reported full vaccination with a COVID-19 mRNA or J&J non-replicating viral vector vaccine (n = 16,020). Exposures: Potential community exposure to SARS-CoV-2. Main Outcome and Measures: Self-reported breakthrough infection. Results: SARS-CoV-2 infection after vaccination was self-reported in 1.9% of participants, with an incidence rate of 7.3 per 100,000 person-years. Younger age (45-64 vs. 18-44: HR (95% CI) = 0.65 (0.51-0.82); 65+ vs. 18-44: HR (95% CI) = 0.59 (0.39-0.90)), and vaccination with J&J Ad26.COV2.S were associated with a higher risk of breakthrough infection compared to vaccination with Pfizer BNT162b2 (Ad26.COV2.S vs. BNT162b2: HR (95% CI) = 2.23 (1.40-3.56)), while participants vaccinated with mRNA-1273 (mRNA-1273 vs. BNT162b2: HR (95% CI) = 0.69 (0.50-0.96) and those residing in urban counties experienced a lower rate of SARS-CoV-2 breakthrough infection compared with those from suburban (HR (95% CI) = 1.39 (1.01-1.90)) or rural (HR (95% CI) = 1.57 (1.16-2.11)) counties. There was no significant association between breakthrough infection and participant sex, race, healthcare worker status, prior COVID-19 infection, routine mask use, or overall vaccination rate in the county of residence. Conclusions and Relevance: This NC community-based observational study showed that the proportion of the cohort who self-report breakthrough SARS-CoV-2 infections was 7.3 events per 100,000 person-years. Younger adults, those vaccinated with J&J Ad26.COV2.S, and those residing in suburban or rural counties were at higher risk of breakthrough infections and should be targeted for additional risk mitigation strategies to decrease community transmission.

Hypothesis: The prevalence of type 2 diabetes is higher in Latino populations compared with other major ancestry groups. Not only has the Latino population been systematically underrepresented in large-scale genetic analyses, but previous studies relied on the imputation of ungenotyped variants based on the 1000 Genomes (1000G) imputation reference panel, which results in suboptimal capture of low-frequency or Latino-enriched variants. The NHLBI Trans-Omics for Precision Medicine (TOPMed) reference panel represents a unique opportunity to analyze rare genetic variations in the Latino population. Methods: We evaluate the TOPMed imputation performance using genotyping array and whole-exome sequence data in 6 Latino cohorts. To evaluate the ability of TOPMed imputation of increasing the identified loci, we performed a Latino type 2 diabetes GWAS meta-analysis in 8,150 type 2 diabetes cases and 10,735 controls and replicated the results in 6 additional cohorts including whole-genome sequence data from the All of Us cohort. Results: We show that, compared to imputation with 1000G, the TOPMed panel improves the identification of rare and low-frequency variants. We identified 26 distinct signals including a novel genome-wide significant variant (minor allele frequency 1.6%, OR=2.0, P=3.4−10-9) near ORC5. A Latino-tailored polygenic score constructed from our data and GWAS data from East Asian and European populations improves the prediction accuracy in a Latino target dataset, explaining up to 7.6% of the type 2 diabetes risk variance. Conclusions: Our results demonstrate the utility of TOPMed imputation for identifying low-frequency variation in understudied populations, leading to the discovery of novel disease associations and the improvement of polygenic scores.