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Tumor suppressor protein p53 is regulated in a number of ways, including during initiation of TP53 mRNA translation. The 50 end of TP53 mRNA contains regulatory structures that enable noncanonical initiation using mechanisms that remain poorly described. Here we analyze per-nucleotide reactivity changes in the 50 end secondary structure of TP53 mRNA under in-cell conditions using A549 human lung carcinoma cells. We first construct a cell-free secondary structure model using SHAPE reagent 5-nitroisatoic anhydride on gently extracted and deproteinated RNA. We observe previously described regulatory features of the TP53 mRNA 50 end including two motifs which we refer to as long stem-loop (LSL) and short stem-loop (SSL), respectively. We observe a domain-forming helix that groups LSL and SSL, forming a three-helix junction. Applying in-cell selective 20 hydroxyl acylation analyzed by primer extension and mutational profiling, we assess reactivity profiles with unstressed cells and with chemically induced stress conditions expected to stimulate TP53 cap-independent translation. We analyze the effects of etoposide-induced DNA damage, CoCl2-induced hypoxia, and 50 cap inhibition with 4EGI-1 treatment. Identifying stress-associated changes in the TP53 50 end may help elucidate therole of regulatory RNA structure in cap-independent translation. Using DSHAPE, we identify in-cell protection sites that correspond with previously described RNA–protein binding sites on the apical loops of LSL and SSL. Furthermore, we identify several other potential interaction sites, some associated with specific types of stress. Some noteworthy changes include DeltaSHAPE sites proximal to the start codons, at the three-helix junction and on the domain-forming helix. We summarize potential interactions on the cell-free secondary structure model. 

What You Will Learn in This Chapter In this chapter, instructors will develop foundational knowledge about how to select and use computational tools to teach CRISPR-Cas technologies. Broadly speaking, CRISPR-Cas is a sequence-based technology. Computational resources provide a platform for managing and interacting with these sequences. With appropriate instructional design, computational tools are a valuable complement to lessons about CRISPR-Cas technologies and are essential support tools for CRISPR-Cas experiments. With an ever-growing suite of computational tools available, in this chapter, instructors will learn to navigate the landscape of these tools to select the most appropriate tools for their classroom or laboratory needs. Instructors will learn to identify when computational resources are appropriate for use in their classroom (and when they are not appropriate), then how to select the most appropriate tools for their unique needs. Additionally, we introduce instructors to best practices in instructional design for using CRISPR-Cas computational tools in the classroom. Throughout, instructors will learn both the rationale and principle behind selection so they can evaluate tools discussed in this chapter and new ones as they become available. 

Abstract Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite. 

The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free L-Trp dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of L-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo L-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. in vitro TnaC-peptidyl-tRNA accumulation and toeprinting assays confirmed that in the presence of L-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that L-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce L-Trp dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of L-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that L-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.