ABSTRACT
Several signals are required for the programmed frameshifting in translation of IS911 mRNA. These include a Shine Dalgarno (SD)-like sequence, a slippery sequence of six adenine residues and a guanine residue (A6G) and a 3' secondary structure. The structure of the mRNA containing these elements was investigated using chemical and enzymatic probing. The probing data show that the 3' structure is a three-way junction of stems. The function of the three-way junction was investigated by mutagenesis. Disrupting the stability of the structure greatly affects frameshifting and transposition levels as tested by separate in vivo assays. Structural probing and thermal melting profiles indicate that the disrupted three-way junctions have altered structures.
Subject(s)
DNA Transposable Elements/genetics , Escherichia coli/genetics , Frameshifting, Ribosomal/genetics , Nucleic Acid Conformation , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid/genetics , Aldehydes/pharmacology , Base Pairing/genetics , Base Sequence , Butanones , CME-Carbodiimide/analogs & derivatives , CME-Carbodiimide/pharmacology , Imidazoles/pharmacology , Molecular Sequence Data , Mutation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Recombination, Genetic/genetics , Ribonucleases/pharmacology , Structure-Activity Relationship , Sulfuric Acid Esters/pharmacology , TemperatureABSTRACT
To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.
