Shine-Dalgarno sequences (SD) in prokaryotic mRNA facilitate protein translation by pairing with rRNA in ribosomes. Although conventionally defined as AG-rich motifs, recent genomic surveys reveal great sequence diversity, questioning how SD functions. Here, we determined the molecular fitness (i.e., translation efficiency) of 4 9 synthetic 9-nt SD genotypes in three distinct mRNA contexts in Escherichia coli. We uncovered generic principles governing the SD fitness landscapes: (1) Guanine contents, rather than canonical SD motifs, best predict the fitness of both synthetic and endogenous SD; (2) the genotype-fitness correlation of SD promotes its evolvability by steadily supplying beneficial mutations across fitness landscapes; and (3) the frequency and magnitude of deleterious mutations increase with background fitness, and adjacent nucleotides in SD show stronger epistasis. Epistasis results from disruption of the continuous base pairing between SD and rRNA. This "chain-breaking" epistasis creates sinkholes in SD fitness landscapes and may profoundly impact the evolution and function of prokaryotic translation initiation and other RNA-mediated processes. Collectively, our work yields functional insights into the SD sequence variation in prokaryotic genomes, identifies a simple design principle to guide bioengineering and bioinformatic analysis of SD, and illuminates the fundamentals of fitness landscapes and molecular evolution.

BACKGROUNDBouveret syndrome is a rare complication of cholelithiasis, with only 315 cases reported in the literature between 1967 and 2016. Delay in diagnosis is associated with a high mortality rate. Diagnosis is based upon clinical manifestations, gastroscopy, and imaging studies such as abdominal computed tomography and magnetic resonance cholan-giopancreatography. Endoscopic stone extraction or lithotripsy is the preferred choice for treatment as it is safe and minimally invasive with few complications. However, if endoscopy fails, surgery is required.CASE SUMMARYA 61-year-old female patient presented with recurrent epigastric pain for more than 6 mo. On endoscopy, a large amount of food residue was present in the stomach with multiple stones and ulcers in the antro-pyloric region. Based on these findings, a diagnosis of gastrolithiasis was made. However, computed tomography of the abdomen revealed the correct diagnosis of Bouveret syndrome. Initially, endoscopic treatment was attempted but it failed. Later, she was successfully managed by cholecystectomy with duodenal stone extraction and fistula repair (one-step method). At the last follow-up 6 mo after surgery, the patient was symptom-free.CONCLUSIONBouveret syndrome is a rare complication of gallstones that requires prompt endoscopic or surgical treatment to prevent mortality.

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Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes.