The bridge α-helix in the β′ subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (β′ 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (β′ 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200 µM NTPs and is strongly reduced in λ tR2 recognition at 1 µM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the β subunit, whereas F773 communicates through the fork domain in the β subunit. I774 interacts with the F-loop, which also contacts the glycine hinge of the bridge helix. These results identified negative and positive circuits coupled at YFI and employed for regulation of catalysis, elongation, termination and translocation.
Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA–DNA hybrid resulting in opening of the furthest upstream i−8 RNA–DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β′ Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ–β-barrel and the associated sandwich barrel hybrid motif (also called the flap domain), the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA–DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K+ and Mg2+ and also an RNA 3′-OH or a 3′-H2 affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K+ and Mg2+concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation.
Retinitis pigmentosa is the most common form of inherited blindness in humans. A well-studied model of the disease is the rd1 mouse, characterized by a loss of function mutation in the catalytic β subunit of the phosphodiesterase 6 (Pde6) holoenzyme involved in phototransduction within rods and cones. The period of photoreceptor degeneration in the rd1 mouse occurs during postnatal days 10–21. In previous work, only Pde6β and vesicular-trafficking protein Prenylated Rab Acceptor 1 (PRA1) have been found to be consistently downregulated during the first ten days following birth. In a yeast-two-hybrid assay conducted by our lab, PRA1 was shown to interact with Charged Multivesicular Body Protein 2B (CHMP2B), an endosomal sorting protein that has been implicated in several neurodegenerative diseases, such as frontotemporal dementia and amyotrophic lateral sclerosis. We investigated whether CHMP2B is mislocalized in the rd1 mouse. Immunohistochemical labeling of CHMP2B was done in both postnatal wild type and rd1 mouse retinas. Prior to the onset of degeneration, CHMP2B immunolabeling was weaker in rd1 retinas, particularly in the developing photoreceptor synaptic layer, compared to wild type. Furthermore, staining of CHMP2B in wild type photoreceptors peaked at postnatal day 12, while CHMP2B staining in rd1 retinas was diffuse and disorganized. In conclusion, these findings show that proper localization of CHMP2B is disrupted in rd1 photoreceptors. Further studies are needed to investigate possible roles for CHMP2B in endocytic activity that is vital to photoreceptor maintenance, as well as differentiation, and development in mouse photoreceptors.
Exonuclease (exo) III was used to probe the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream and upstream borders. In the absence of NTPs, RNAP appears to stall primarily in a post‐translocated state and to return slowly to a pre‐translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation. The forward translocation state is made more stable by elevating the KCl and Mg2+ concentrations into the physiological range (150 versus 40 mM KCl; 5 versus 0.5 mM Mg2+), indicating that, under natural conditions, translocation tends to stick in the post‐translocated position. RNAP mutants in the N‐terminal bridge helix hinge beta’ 778‐GARKGL‐783 strongly affect TEC translocation, NTP binding and stability. Mutations that stiffen the hinge (i.e. GARKGL‐>AARKAL) are strongly defective for holding the forward translocation register and for stable NTP binding but do not destabilize the TEC. Reminiscent of a ball and socket joint, the bulky R780 is surrounded by the beta fork, when the bridge helix is in a relatively straight conformation. An overly flexible hinge (i.e. K781A), however, may result in misaligning R780 within the fork resulting in low transcriptional activity. Molecular dynamics simulation indicates that the GARKGL hinge can completely unwind, but only if the beta’ trigger loop is in an open conformation. Grant Funding Source: NSF and NIH
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