Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry and product release. The non-covalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting non-cognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule Förster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations - open, closed and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3′-5′ exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair.
The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.
Recent observations of RNA interference (RNAi) in the nuclei of human cells raise key questions about the extent to which nuclear and cytoplasmic RNAi pathways are shared. By directly visualizing the localization of small interfering RNA (siRNA) in live human cells, we show here that siRNA either selectively localizes in the cytoplasm or translocates into the nucleus, depending on where the silencing target RNA resides. Two siRNAs that target the small nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In contrast, an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA dissociates, and only antisense strand distributes in the cytoplasm of the cells harboring the target RNA, whereas sense strand gets degraded. At the same time, both strands of the latter siRNA are distributed throughout the cytoplasm and nucleus in cells lacking the silencing target RNA. These results suggest the existence of a mechanism by which the RNAi machinery orchestrates a target-determined localization of the siRNA and the corresponding RNAi activity, and also provide evidence for formation of nuclear-programmed active RNA induced silencing complexes directly in the nucleus.confocal imaging ͉ nuclear͞cytoplasmic localization ͉ RNA-induced silencing complex ͉ RNA interference mechanism ͉ small interfering RNA N early a decade after the discovery that double-stranded RNA can trigger an RNAi response that inhibits gene expression in a sequence-specific manner (1), the complexity of the mechanisms by which small RNAs regulate gene expression continues to unfold (2-11). RNA interference (RNAi) has generally been defined as a cellular pathway that mediates posttranscriptional gene silencing either by sequence-specific degradation of targeted RNAs or via sequence-specific inhibition of translation. Thus, RNAi studies in mammalian cells have mainly focused on the cytoplasm, where mature mRNA is translated and key proteins of RNA-induced silencing complexes (RISCs) were thought to localize and function. These RISCs, by which the RNAi machinery implements silencing of gene expression, are composed of several proteins (including Ago1 and Ago2) and one strand of small interfering RNA (siRNA) (12, 13). During the course of RISC assembly, the siRNA͞ microRNA duplex dissociates, and the guide strand enters active RISCs, allowing binding and degradation of the complementary target mRNA.Target specificity in RNAi is achieved through RNA-RNA sequence recognition and base pairing. Because RNA can also recognize and form duplexes with DNA, RNAi should be capable of affecting gene function at the level of genomic DNA, extending the realm of RNAi function into the nucleus. Indeed, recent demonstrations of siRNA-induced transcriptional gene silencing through involvement of DNA methylation (2, 3) in various human cell types, siRNA-dependent knock-down of nucleus-restricted transcripts (4, 5), and a direct documentation of potent and specific down-regulation of 7SK and U6 small nuclear RNAs (6) have uncovered such nuclear RNAi pathways in ...
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