Single-mode termination of phage transcriptions, disclosing bacterial adaptation for facilitated reinitiations.

Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.