ABSTRACT
Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.
Subject(s)
Bacteroidetes/genetics , Peptide Chain Initiation, Translational , Regulatory Sequences, Ribonucleic Acid , Ribosomes/metabolism , Amino Acid Sequence , Base Sequence , Codon, Initiator , Cryoelectron Microscopy , Crystallography, X-Ray , Escherichia coli/genetics , Flavobacterium/genetics , Gene Expression Regulation, Bacterial , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Puromycin/pharmacology , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , RNA, Ribosomal, 5S/genetics , Ribosomes/ultrastructure , Sequence Alignment , Sequence Homology , Species SpecificityABSTRACT
In retroviruses, the Gag protein is a precursor from which the mature proteins matrix, capsid, and nucleocapsid are derived. Gag plays an important structural role in the assembly of virions at the plasma membrane. While Gag proteins from several different retroviruses have been purified for study in vitro, there has yet to be a report of successful purification of deltaretroviral Gag. In this paper, we report the cloning, expression and purification of full-length bovine leukemia virus (BLV) Gag from Escherichia coli using a combination of polyethyleneimine precipitation, ammonium sulfate precipitation, and affinity chromatography. Experiments using size-exclusion chromatography were also performed to analyze the oligomeric state of the Gag protein in solution, and results suggest that it exists primarily as a monomer but may oligomerize into higher-order complexes to a small extent. Molecular weight estimation by comparison of elution volume to a set of protein standards supports the hypothesis that BLV Gag adopts a slightly extended conformation in solution. The results are discussed in comparison to the solution structure and assembly pathways of other retrovirus genera.