Structural and genetic studies of stimulators for recoding;
Recoding is the programmed alteration of translation, a dynamic process in which specific signals within the mRNA act as stimulators to alter the ribosomes reading of the genetic code. There are three broad classes of recoding including translational frameshifting, redefinition of codons, and translational bypass. In frameshifting the translational machinery acts on signals from stimulators contained in the mRNA to shift into the -1 or +1 frame. Redefinition of codon meaning is a specific case in which the stop codons UAG or UGA are used to code for an amino acid and, as with frameshifting, signals found within the mRNA direct the recoding event. Translational bypass is defined by the skipping of relatively long stretches of a gene. Again, signals found within the mRNA direct the bypass event. Stimulators for recoding events include elements located either 3' or 5' of the recoding site. These signals include but are not limited to RNA secondary structures, Shine-Dalgamo sequences, stop codons, and the nascent polypeptide chain. In this study we have used structural and genetic methods to investigate the stimulatory signals involved in two recoding events: the -1 frameshifting in bacterial insertion sequence IS977 and the translational bypass in bacteriophage T4 gene<50. Several signals are required for the programmed frameshifting in translation of IS977 mRNA. These include a Shine Dalgamo (SD)-like sequence, a slippery sequence of six adenines and a guanine, A AAA AAG, (6 AG) 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. In the translational bypass of bacteriophage T4 gene60 the stimulatory signals include: (1) the matched GGA take-off and landing site codons; (2) a UAG stop codon just 3' of the take-off site; (3) a portion of the nascent peptide composed of charged, polar, and hydrophobic residues; (4) a RNA secondary structure found in the skipped message (coding gap); and (5) a GAG sequence found 6 nucleotides from the landing site that acts as a Shine-Dalgamo sequence. The sequence of the mRNA containing the secondary structure was investigated using chemical and enzymatic probing, nuclear magnetic resonance (NMR), and site specific mutagenesis. The probing and NMR data show that the mRNA secondary structure is composed of at least two stem-loop structures. The function of the extended structure was tested using site specific mutagenesis and it was found that disrupting the second stem-loop significantly affects bypassing levels as tested by two separate assays. The Shine-Dalgamo sequence was also investigated using site specific mutagenesis, specialized ribosomes and mass spectrometry. Here we present evidence that the GAG sequence acts as a Shine-Dalgamo interaction to promote landing site efficiency and fidelity. We have found that altering this sequence to create either a stronger or weaker Shine-Dalgamo interaction reduces overall bypass efficiency when tested using reporter systems. Furthermore, using a specialized ribosome system we have shown that ribosomes with a mutated anti-Shine-Dalgamo sequence can rescue bypass efficiency of mutant genedO sequences containing altered GAG stimulatory sequences. Finally, we have shown that when landing sites are engineered into the coding gap, ribosomes can land at these sites at the expense of landing at the downstream wild-type landing site. The efficiency of the landing at these sites is greatly improved when the alternate GGA landing site is in tandem with the stimulatory GAG stimulatory sequences. Finally, we have shown that when landing sites are engineered into the coding gap, ribosomes can land at these sites at the expense of landing at the downstream wild-type landing site. The efficiency of the landing at these sites is greatly improved when the alternate GGA landing site is in tandem with the stimulatory GAG sequence. We also present data for a new set of vectors that will aid in the study of landing site fidelity. We have produced a vector, plasmid pNTH, that is a fusion of the E. coli NusA gene with gene60 and a unique three-frame six-histidine (TRIHIS) sequence that allows for the isolation of bypass protein products from all three frames. Control constructs showed that proteins produced by landing in all three frames could be easily purified and analyzed using mass spectrometry (MS). Mutant gene60 sequences with a UCC take-off codon and multiple UCC landing sites were produced to test for landing site fidelity in this system. In all of these constructs landing was at the w.t. location indicating that the gene60 bypass signals direct the ribosome to land at the correct site. Gene60 constructs containing AAA take-off sites and either a 12 A or 6 A landing site were tested. In this construct ribosomes were able to land in two or three frames. These data show that we have developed a powerful system for further analysis of landing site fidelity, and that landing site choice in the w.t. context is very accurate.