The programmed −1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well
conserved structured RNA found in all coronaviruses’ genomes. By adopting a
pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal
frameshifting event near the stop codon of the first open reading frame Orf1a during
translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a
new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins
pp1a and pp1ab produce nonstructural proteins NSPs 1–10 and NSPs 1–16,
respectively, which contribute vital functions during the viral life cycle and must be
present in the proper stoichiometry. Both drugs and sequence alterations that affect the
stability of the −1 programmed ribosomal frameshifting element disrupt the
stoichiometry of the NSPs produced, which compromise viral replication. For this reason,
the −1 programmed frameshifting element is considered a promising drug target.
Using chaperone assisted RNA crystallography, we successfully crystallized and solved
the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot
structure with the three stems stacked in a vertical orientation stabilized by two
triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure
provides a new conformation of PFSE distinct from the bent conformations inferred from
midresolution cryo-EM models and provides a high-resolution framework for mechanistic
investigations and structure-based drug design.
Methicillin resistant Staphylococcus aureus (MRSA) is a
public health threat worldwide. Although the mobile genomic island responsible
for this phenotype, called SCC, was labeled non-replicative, we predicted DNA
replication-related functions for some of their conserved proteins. We show that
one of these, Cch, is homologous to the self-loading initiator helicases of an
unrelated family of genomic islands, that it is an active 3’ to
5' helicase, and that the adjacent ORF encodes an ssDNA-binding protein.
Our 2.9Å crystal structure of intact Cch shows that it forms a hexameric
ring. Cch belongs to the pre-sensor II insert clade of AAA+ ATPases, as do the
archaeal and eukaryotic MCM-family replicative helicases. Additionally, we find
that SCC elements are part of a broader family of mobile elements that all
encode a replication initiator upstream of their recombinases. Replication after
excision would enhance the efficiency of horizontal gene transfer.
Sirtuins were originally shown to regulate a wide array of biological processes such as transcription, genomic stability, and metabolism by catalyzing the NAD+-dependent deacetylation of lysine residues. Recent proteomic studies have revealed a much wider array of lysine acyl modifications in vivo than was previously known, which has prompted a reevaluation of sirtuin substrate specificity. Several sirtuins have now been shown to preferentially remove propionyl, succinyl, and long-chain fatty acyl groups from lysines, which has changed our understanding of sirtuin biology. In light of these developments, we revisited the acyl specificity of several well-studied archaeal and bacterial sirtuins. We find that the Archaeoglobus fulgidus sirtuins, Sir2Af1 and Sir2Af2, preferentially remove succinyl and myristoyl groups, respectively. Crystal structures of Sir2Af1 bound to a succinylated peptide and Sir2Af2 bound to a myristoylated peptide show how the active site of each enzyme accommodates a noncanonical acyl chain. As compared to its structure in complex with an acetylated peptide, Sir2Af2 undergoes a conformational change that expands the active site to accommodate the myristoyl group. These findings point to both structural and biochemical plasticity in sirtuin active sites and provide further evidence that sirtuins from all three domains of life catalyze noncanonical deacylation.PDB Code(s): 4TWI; 4TWJ
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