Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5′-Untranslated Region of SARS-CoV-2

Throughout the family of coronaviruses, structured RNA elements within the 5 ' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b. We herein report the detailed characterization of the structure and dynamics of elements SL5a and SL5b that are located immediately upstream of the SARS-CoV-2 ORF1a/b start codon. Exploiting the unique ability of solution NMR methods, we show that the structures of both apical loops are modulated by structural differences in the remote parts located in their stem regions. We further integrated our high-resolution models of SL5a/b into the context of full-length 5_SL5 structures by combining different structural biology methods. Finally, we evaluated the impact of the two most common VoC mutations within 5_SL5 with respect to individual base-pair stability.