In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state

It has been known for decades that purified small subunits of the ribosome can interconvert between active and inactive conformations in experiments performed under simplified conditions, but the physiological relevance of this switch has remained unclear. We probed the structure of ribosomal RNA in healthy living cells and discovered that stably assembled 30S subunits exist predominantly in the inactive conformation, with structural differences localized in the functionally important decoding region. Disrupting the ability to interconvert between active and inactive conformations compromised translation in cells. In-cell RNA structure probing supports a model in which “inactive” 30S subunits comprise an abundant in-cell state that regulates ribosome function

An inclusive Research and Education Community (iREC) model to facilitate undergraduate science education reform

Funding: This work was supported by Howard Hughes Medical Institute grants to DIH is GT12052 and MJG is GT15338.Over the last two decades, there have been numerous initiatives to improve undergraduate student outcomes in STEM. One model for scalable reform is the inclusive Research Education Community (iREC). In an iREC, STEM faculty from colleges and universities across the nation are supported to adopt and sustainably implement course-based research – a form of science pedagogy that enhances student learning and persistence in science. In this study, we used pathway modeling to develop a qualitative description that explicates the HHMI Science Education Alliance (SEA) iREC as a model for facilitating the successful adoption and continued advancement of new curricular content and pedagogy. In particular, outcomes that faculty realize through their participation in the SEA iREC were identified, organized by time, and functionally linked. The resulting pathway model was then revised and refined based on several rounds of feedback from over 100 faculty members in the SEA iREC who participated in the study. Our results show that in an iREC, STEM faculty organized as a long-standing community of practice leverage one another, outside expertise, and data to adopt, implement, and iteratively advance their pedagogy. The opportunity to collaborate in this manner and, additionally, to be recognized for pedagogical contributions sustainably engages STEM faculty in the advancement of their pedagogy. Here, we present a detailed pathway model of SEA that, together with underpinning features of an iREC identified in this study, offers a framework to facilitate transformations in undergraduate science education.Peer reviewe

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection