We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single... more We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, l exonuclease, RNAse T1, and treatment with KMnO 4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5 H to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the ®ngers subdomain of the RNAP to clamp down on the downstream end of the template strand.
The role of steric constraints vs sequence preference in start site selection by T7 RNA polymeras... more The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand 'CC' initiation sequence was moved away from its normal position relative to the-17 to-6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription.
We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA... more We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modi®cations on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.
The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extrace... more The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(oaminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentrationand time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (V max) but no change in K m. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the V max of DA transport without altering K m. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.
F1000 - Post-publication peer review of the biomedical literature, 2007
Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of ... more Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is Synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phospho-inositol phosphatase domains and a proline-rich domain. Genetic ablation of Synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle re-availability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of Synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1−/− neurons. Our studies show that Synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and post-endocytic vesicle re-availability can be fully restored upon reintroduction of Synaptojanin1. However, expression of Synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and re-availability.
We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single... more We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, l exonuclease, RNAse T1, and treatment with KMnO 4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5 H to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the ®ngers subdomain of the RNAP to clamp down on the downstream end of the template strand.
The role of steric constraints vs sequence preference in start site selection by T7 RNA polymeras... more The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand 'CC' initiation sequence was moved away from its normal position relative to the-17 to-6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription.
We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA... more We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modi®cations on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.
The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extrace... more The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(oaminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentrationand time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (V max) but no change in K m. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the V max of DA transport without altering K m. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.
F1000 - Post-publication peer review of the biomedical literature, 2007
Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of ... more Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is Synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phospho-inositol phosphatase domains and a proline-rich domain. Genetic ablation of Synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle re-availability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of Synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1−/− neurons. Our studies show that Synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and post-endocytic vesicle re-availability can be fully restored upon reintroduction of Synaptojanin1. However, expression of Synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and re-availability.
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Papers by Eileen Lafer