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Eckhard Jankowsky

Publications and source records attributed to Eckhard Jankowsky.

14 recordsLinked to original sources
PubMed2006

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases.

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.

DEAD-box RNA Helicases↗
PubMed2006

Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

Adenosine Triphosphate↗
PubMed2006

Remodeling of ribonucleoprotein complexes with DExH/D RNA helicases.

The DExH/D protein family is the largest group of enzymes in eukaryotic RNA metabolism. DExH/D proteins are mainly known for their ability to unwind RNA duplexes in an ATP-dependent fashion. However, it has become clear in recent years that these DExH/D RNA helicases are also involved in the ATP-dependent remodeling of RNA-protein complexes. Here we review recent studies that highlight physiological roles of DExH/D proteins in the displacement of proteins from RNA. We further discuss work with simple RNA-protein complexes in vitro, which illuminates mechanisms by which DExH/D proteins remove proteins from RNA. Although we are only beginning to understand how DExH/D proteins remodel RNA-protein complexes, these studies have shown that an 'RNA helicase' does not per se require cofactors to displace proteins from RNA, that protein displacement does not necessarily involve RNA duplex unwinding, and that not all DExH/D proteins are able to disassemble the same range of ribonucleoproteins.

Animals↗
PubMed2006

Discriminatory RNP remodeling by the DEAD-box protein DED1.

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the "inability" to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

Adenosine Triphosphate↗
PubMed2006

Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track.

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.

Base Sequence↗
PubMed2005

ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1.

DEAD-box RNA helicases, which are involved in virtually all aspects of RNA metabolism, are generally viewed as enzymes that unwind RNA duplexes or disrupt RNA-protein interactions in an ATP-dependent manner. Here, we show in vitro that the DEAD-box protein DED1 from Saccharomyces cerevisiae promotes not only RNA unwinding but also strand annealing, the latter in such a profound fashion that the physical limit for a bimolecular association rate constant is approached. We further demonstrate that DED1 establishes an ATP-dependent steady state between unwinding and annealing, which enables the enzyme to modulate the balance between the two opposing activities through ATP and ADP concentrations. The ratio between unwinding and annealing and the degree to which both activities are ATP- and ADP-modulated are strongly influenced by structured as well as unstructured regions in the RNA substrate. Collectively, these findings expand the known functional repertoire of DEAD-box proteins and reveal the capacity of DED1 to remodel RNA in response to ADP and ATP concentrations by facilitating not only disruption but also formation of RNA duplexes.

Adenosine Diphosphate↗
PubMed2005

Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation.

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.

Animals↗
PubMed2005

Prediction of functional tertiary interactions and intermolecular interfaces from primary sequence data.

Given the availability of sequence information for many species, one can examine how the sequence of a gene varies among different organisms. This is accomplished by aligning the sequences and observing patterns of conservation, mutation and counter-mutation at different positions in the gene. Imbedded in these patterns is information on energetic coupling and macromolecular interactions, which can be deciphered by application of statistical algorithms. Here we report a robust approach for predicting interactions within (or between) any type of biopolymer, including proteins, RNAs and RNA-protein complexes. Rather than maximize the number of predictions, this approach is designed to detect a limited number of highly significant interactions, thereby providing accurate results from alignments that contain a modest number of sequences (20-60). The versatility and accuracy of the algorithm is demonstrated by the successful prediction of important intramolecular interactions within RNAs, modified RNAs, and proteins, as well as the prediction of RNA-protein and protein-protein interactions.

Algorithms↗
PubMed2005

RNA helicases: versatile ATP-driven nanomotors.

RNA helicases are a large family of molecular motors that utilize nucleoside triphosphates to unwind RNA duplexes and to remodel RNA protein complexes. In this review, we discuss the structure and function of RNA helicases with an emphasis on the potential application of these enzymes to control conformational changes in nanoassemblies that contain RNA.

Adenosine Triphosphate↗
PubMed2004

Backbone tracking by the SF2 helicase NPH-II.

Members of the DExH/D family of proteins, a subset of helicase superfamily 2 (SF2), are involved in virtually all aspects of RNA metabolism. NPH-II, a prototypical member of this protein family, exhibits robust RNA helicase activity in vitro. Using a series of modified substrates to explore the unwinding mechanism of NPH-II, we observed that the helicase tracks exclusively on the loading strand, where it requires covalent continuity and specifically recognizes the ribose-phosphate backbone. NPH-II unwinding was unaffected by lesions and nicks on the top strand, which has a minimal role in substrate recognition. NPH-II required physical continuity of phosphodiester linkages on the loading strand, although abasic regions were tolerated. These findings suggest that SF2 helicases are mechanistically distinct from other helicase families that can tolerate breaks in the loading strand and for which bases are the primary recognition determinant.

Binding Sites↗
PubMed2004

Protein displacement by DExH/D "RNA helicases" without duplex unwinding.

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes but can be exerted on a wide range of ribonucleoprotein substrates.

Adenosine Triphosphate↗
PubMed2002

The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding.

The RNA helicase/protease NS3 plays a central role in the RNA replication of hepatitis C virus (HCV), a cytoplasmic RNA virus that represents a major worldwide health problem. NS3 is, therefore, an important drug target in the effort to combat HCV. Most work has focused on the protease, rather than the helicase, activities of the enzyme. In order to further characterize NS3 helicase activity, we evaluated individual stages of duplex unwinding by NS3 alone and in complex with cofactor NS4A. Despite a putative replicative role in RNA unwinding, we found that NS3 alone is a surprisingly poor helicase on RNA, but that RNA activity is promoted by cofactor NS4A. In contrast, NS3 alone is a highly processive helicase on DNA. Phylogenetic analysis suggests that this robust DNA helicase activity is not vestigial and may have specifically evolved in HCV. Given that HCV has no replicative DNA intermediate, these findings suggest that NS3 may have the capacity to affect host DNA.

Base Sequence↗
PubMed2002

mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.

Human melanoma cells can be reprogrammed to terminally differentiate and irreversibly lose proliferative capacity by appropriate pharmacological manipulation. Subtraction hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversion, and programmed cell death (apoptosis). This gene contains both a caspase recruitment domain and putative DExH group RNA helicase domains. Atypical helicase motifs of MDA-5 deviate from consensus sequences but are well conserved in a potentially new group of cloned and hypothetical proteins. mda-5 is an early response gene inducible by IFN and tumor necrosis factor-alpha, responding predominantly to IFN-beta. Protein kinase C activation by mezerein further augments mda-5 expression induced by IFN-beta. Expression of mda-5 is controlled transcriptionally by IFN-beta, and the MDA-5 protein localizes in the cytoplasm. mda-5 displays RNA-dependent ATPase activity, and ectopic expression of mda-5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase that contains both a caspase recruitment domain and RNA helicase motifs, with a confirmed association with growth and differentiation in human melanoma cells.

Adenosine Triphosphatases↗