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The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro . RNA tags in the Escherichia coli large ribosomal (50S) subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes. 

Abstract The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3′-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts. 

A persistent problem in engineering is an insufficient number of students interested in pursuing engineering as a college major and career. Middle school is a critical time where student interest, identity, and career choices begin to solidify. Student interest in engineering at the K-12 level has been shown to predict whether they pursue engineering as a college major and career. Therefore, research is needed to determine if engineering summer camp activities affect engineering interest and identity in middle school students and in this paper, we present a research study approach to achieve the stated objective. To develop engineering-specific theories of how engineers are formed, this paper explores interest and identity development of three middle-school populations participating in engineering summer camps offered by the College of Engineering at a Western land-grant institution: (1) Young women in engineering camp (2) First generation camp and, (3) Introduction to engineering camp. The camps are identical in content and designed with the goal of increasing understanding of different engineering fields and careers. The only difference between the three camps is that the women-focused and first generation camps involve participation of guest speakers and role-model mentors appropriate for the camp populations. The main objective of designing this mixed-methods research study is to answer three research questions: (1) How strongly are engineering identity and interest linked to the intention to pursue engineering as a major in college and as a future career? (2) Which specific activities in the camps lead to a change in identity and interest in engineering? (3) To what extent and in what ways do the qualitative participant focus group interviews and observations of participants engaged in camp activities addressing research question (2) contribute to a comprehensive understanding of the quantitative data obtained via pre- and post-surveys addressing research question (1)? The research design leverages existing quantitative surveys. Additionally, focus groups and observations will be based on a selected set of questions from these surveys. The research design consists of one phase with two data streams. Quantitative data are gathered in Phase 1 from two data collection points: first, when students register for the camp and, second, at the end of the camp (post-survey). Qualitative data in the form of in-depth focus group interviews (at the end of the camp) with 4 – 5 participants per focus group and observations of camp activities during the five days of camp are implemented. For the qualitative analysis, Grounded Theory is utilized for analyzing focus group interview and observation transcripts using an iterative process that involves reading, discussing, and coding. This paper will present details of the quantitative and qualitative analysis methods used for this study. The research is funded by the National Science Foundation PFE:RIEF program. 

Abstract In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.