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Biomedical subjects

E Jankowsky

Publications and source records attributed to E Jankowsky.

9 recordsLinked to original sources
PubMed2001

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase.

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.

3' Untranslated Regions↗
PubMed2000

The DExH protein NPH-II is a processive and directional motor for unwinding RNA.

All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion. DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics. All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA. Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies. Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown. Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn. We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA.

Kinetics↗
PubMed2000

The DExH/D protein family database.

DExH/D proteins are essential for all aspects of cellular RNA metabolism and processing, in the replication of many viruses and in DNA replication. DExH/D proteins are subject to current biological, biochemical and biophysical research which provides a continuous wealth of data. The DExH/D protein family database compiles this information and makes it available over the WWW (http://www.columbia.edu/ ej67/dbhome.htm ). The database can be fully searched by text based queries, facilitating fast access to specific information about this important class of enzymes.

Amino Acid Sequence↗
PubMed1998

Oligonucleotide facilitators enable a hammerhead ribozyme to cleave long RNA substrates with multiple-turnover activity.

Trans-acting hammerhead ribozymes are usually efficient in cleaving short RNA model substrates under both single-turnover and multiple-turnover conditions. In contrast, when long RNAs are the substrates, the cleavage efficiency of these ribozymes decreases, including a loss of multiple-turnover activity in many cases. Since target substrates for potential therapeutical purposes are mostly long RNAs, a multiple-turnover cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facilitators, capable of enhancing multiple-turnover activity with short substrates, can also affect or cause multiple turnover with long substrates. We examined the effects of 12-base and 24-base oligonucleotide facilitators on the multiple-turnover activity with substrates of different length containing 39-, 452- and 942-base sequences of the human tissue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cleaved only the 39-base substrate with multiple-turnover activity, but not the long 452-base and 942-base substrates. However, facilitator addition enabled the ribozyme to cleave even the 452-base and the 942-base substrates with multiple-turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates. The data demonstrate that a hammerhead ribozyme which, by itself, can only act as a single-turnover catalyst with long substrates, can be switched by facilitators into a multiple-turnover catalyst. Thus, the inactivation of long target RNAs in multiple-turnover reactions may be achieved by addition of oligonucleotide facilitators.

Humans↗
PubMed1998

The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.

Adenosine Triphosphatases↗
PubMed1997

Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes.

Humans↗
PubMed1996

Efficient improvement of hammerhead ribozyme mediated cleavage of long substrates by oligonucleotide facilitators.

Hammerhead ribozymes were found to be not very efficient in cleaving long RNA substrates in trans. Oligonucleotide facilitators, capable of affecting hammerhead ribozymes by interacting with the substrate at the termini of the ribozyme, may improve this reaction. We determined in vitro the effects of 18 DNA and RNA oligonucleotide facilitators on three substrates containing 39, 452, and 942 nucleotides, respectively, by estimating the facilitator influences on association between ribozyme and substrate and on the cleavage step. The effects increase with the length of the substrates. With the 39mer substrate a maximal 4-fold enhancement of the ribozyme activity could be detected, the reaction with the 942mer substrate was accelerated up to 115-fold by facilitator addition. In long, structured substrates the facilitators have the potential to preform the substrate for the ribozyme attack. Due to this preforming effect, the rate of ribozyme-substrate association was increased as well as the rate of the cleavage step. 3'-End facilitators accelerate both of these rates, largely independent on the facilitator length. The rate of the cleavage step is raised as a result of a favorable activation energy gain by these facilitators. With all substrates, the 5'-end facilitators increase the association rate between ribozyme and substrate in dependence on their length. With the 39mer substrate the 5'-end facilitators decrease the rate of the cleavage step. With the long substrates 5'-end facilitators partially increase the rate of the cleavage step due to their preforming potential with these substrates. In some examples, combinations of several 5'-end and 3'-end facilitators provide an additional improvement over single facilitators in both the association between ribozyme and substrate and the cleavage step. Results suggest that even short facilitators may be efficient effectors enhancing hammerhead ribozyme mediated cleavage of long substrates.

Base Sequence↗
PubMed1996

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems.

Base Sequence↗