SUMMARY Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass 50 nucleotides in its middle, resuming translation 3’ of this region. How this large-scale, specific hop occurs, and what determines whether a ribosome bypasses, remains unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans the mRNA in search of the optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements.

Our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. We used the ab initio frameshift prediction program, GeneTack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. We grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position of the frameshift and its direction. We identified 4010 pseudogene clusters and 146 clusters of fs-genes apparently using recoding (local deviation from using standard genetic code) due to possessing specific sequence motifs near frameshift positions. Particularly interesting was finding of a novel type of organization of the dnaX gene, where recoding is required for synthesis of the longer subunit, τ. We selected 20 clusters of predicted recoding candidates and designed a series of genetic constructs with a reporter gene or affinity tag whose expression would require a frameshift event. Expression of the constructs in Escherichia coli demonstrated enrichment of the set of candidates with sequences that trigger genuine programmed ribosomal frameshifting; we have experimentally confirmed four new families of programmed frameshifts.

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3′ adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3′ nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.transcriptional realignment | stem loop stimulator | heteropolymeric slippage-prone motifs | frameshifting A relatively neglected aspect of gene expression is functionally important RNA-DNA hybrid realignment within the RNA polymerase (RNAP) transcribing coding sequence. Such realigned polymerases yield transcripts lacking, or containing, one or more additional base(s) corresponding to the slippage-prone sequence. The fraction of the mRNA population containing deletions or insertions with respect to the template that are not of 3 nucleotides (nt) or multiples thereof, yields proteins that are either truncated, have C-terminal regions not encoded by the zero frame of the template DNA, or both. Despite the limited investigations, several diverse functional slippage-derived products have already been characterized.Bacterial multisubunit DNA-dependent RNAPs undergo slippage on homopolymeric runs of As or Ts. Transcriptional slippage on a run of nine As in the Thermus thermophilus dnaX gene results in heterogeneous transcripts that lead to 50% of the ribosomes quickly terminating. This termination yields a DNA polymerase subunit that lacks C-terminal domains and is synthesized in a 1:1 ratio with the full-length product (1). In other bacteria, counterpart slippage is required for synthesis of the fulllength rather than the truncat...

No abstract