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S R Green

Publications and source records attributed to S R Green.

At least 37 records · Page 2Linked to original sources
PubMed1995

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

Amino Acid Sequence↗
PubMed1995

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.

Amino Acid Sequence↗
PubMed1994

Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain.

The interferon-induced, dsRNA-activated human protein kinase (PKR) exerts antiviral and antiproliferative effects through inhibition of protein synthesis. Studies of structure-function relationships in PKR have shown that two dsRNA binding motifs are important for its autophosphorylation and activation by dsRNA in vitro. To correlate these findings with the activity of PKR in vivo, we examined the function of various PKR deletion mutants in cultured cells by using an inducible expression system. In a reporter gene assay, mutant forms of the kinase lacking amino acids 1-97 (delta 1-97) and 104-157 (delta 104-157), which are required for dsRNA binding in vitro, retained full activity in vivo. Deletion of amino acids 233-271 (delta 233-271), however, abolished the translational inhibitory activity of the kinase and prevented its phosphorylation. Moreover, cells infected with vaccinia virus recombinants expressing wild-type PKR, the mutant delta 104-157, delta 186-222), developed almost complete inhibition of both viral and cellular protein synthesis was upon induction of PKR. This inhibition of viral protein synthesis was not observed in cells infected with a recombinant expressing delta 233-271 mutant PKR. Our findings establish that the region encompassing amino acids 233-271 of PKR is critical for kinase activity in vivo, whereas its dsRNA binding domain is dispensable.

Animals↗
PubMed1993

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells.

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours post-infection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Adenoviruses, Human↗
PubMed1993

Lean and serene.

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Dental Staff↗
PubMed1992

Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function.

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis.

Base Sequence↗
PubMed1992

Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI.

The protein kinase DAI, the double-stranded RNA-activated inhibitor of translation, is an essential component of the interferon-induced cellular antiviral response. The enzyme is regulated by the binding of activator and inhibitor RNAs. We synthesized DAI in vitro and located its RNA-binding domain within the amino-terminal 171 residues. This domain contains two copies of an RNA-binding motif characterized by a high density of basic amino acids, by the presence of conserved residues, and by a probable alpha-helical structure. Deletion of either of the two motifs prevents the binding of dsRNA, but their relative positions can be exchanged, suggesting that they cooperate to interact with dsRNA. Clustered point mutations within the RNA-binding motifs and duplications of the individual motifs indicate that the first copy of the motif plays the more important role. Mutations that impair binding have similar effects on the binding of double-stranded RNAs of various lengths and of adenovirus VA RNAI, implying that discrimination between activator and inhibitory RNAs takes place subsequent to RNA binding.

Amino Acid Sequence↗
PubMed1992

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

Binding Sites↗
PubMed1991

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.

Eukaryotic Initiation Factor-2↗
PubMed1991

Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha.

Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha). The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha. Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa). Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.

Amino Acid Sequence↗
PubMed1991

Selective uptake of cholesteryl esters from low density lipoproteins in vitro and in vivo.

Evidence for the direct uptake ("selective uptake") of cholesteryl esters (CE) from low density lipoproteins (LDL) by perfused luteinized rat ovaries (Azhar, S., A. Cooper, L. Tsai, W. Maffe, and E. Reaven. 1988. J. Lipid Res. 29: 869-882) led to this examination of LDL selective uptake in cultured cells and in rats using LDL doubly labeled with intracellularly trapped tracers of the CE and apoB moieties. Studies in vitro demonstrated LDL selective uptake by human fibroblasts at a low rate relative to LDL particle uptake; the fractional rate of this selective uptake increased with decreasing LDL particle size. Mouse Y1-BS1 adrenal cortical tumor cells also selectively took up LDL CE; on ACTH treatment, LDL selective uptake increased in parallel with high density lipoproteins (HDL) selective uptake, and accounted for the majority of LDL CE uptake. Metabolism of doubly labeled LDL was examined in rats. Adrenal gland and liver selectively took up CE from rat LDL, as did lung and adipose tissue. Selective uptake from human LDL was at a lower fractional rate than from rat LDL, and could not be demonstrated in as many organs. Although selective uptake from LDL by ovaries of adult rats was not significant, ovaries of immature rats consistently exhibited LDL selective uptake; on treatment of these rats with hormones to produce superovulated, luteinized ovaries, LDL selective uptake increased in the ovaries and nowhere else. Selective uptake was also apparent in liver, where it accounted for 27% of total hepatic uptake of rat LDL CE. These studies indicate a significant contribution of selective uptake to LDL CE metabolism in rats, suggesting the possibility of a role in other animals as well.

Adrenocorticotropic Hormone↗
PubMed1991

Comparative acyl specificities for transfer and selective uptake of high density lipoprotein cholesteryl esters.

This study compares the specificities of selective uptake and transfer mediated by plasma cholesteryl ester transfer protein (CETP) for various species of cholesteryl esters in high density lipoproteins (HDL). [3H]Cholesterol was esterified with a series of variable chain length saturated acids and a series of variably unsaturated 18-carbon acids. These were incorporated into synthetic HDL particles along with 125I-labeled apoA-I as a tracer of HDL particles and [14C]cholesteryl oleate as an internal standard for normalization between preparations. Selective uptake by Y1-BS1 mouse adrenal cortical tumor cells was most extensively studied, but uptake by human HepG2 hepatoma cells and fibroblasts of human, rat, and rabbit origin were also examined. Acyl chain specificities for selective uptake and for CETP-mediated transfer were conversely related; selective uptake by all cell types decreased with increasing acyl chain length and increased with the extent of unsaturation of C18 chains. In contrast, CETP-mediated transfer increased with acyl chain length, and decreased with unsaturation of C18 chains. The specificities of human and rabbit CETP were also compared, and were found to differ little. Associated experiments showed that HDL-associated triglycerides, traced by [3H]glyceryl trioleyl ether, were selectively taken up but at a lesser rate than cholesteryl esters. The mechanism of this uptake appears to be the same as for selective uptake of cholesteryl esters.

Acylation↗
PubMed1989

Cholesteryl oleyl and linoleyl ethers do not trace their ester counterparts in animals with plasma cholesteryl ester transfer activity.

Cholesteryl ethers are nonhydrolyzable tracers of cholesteryl esters. We report here that the ethers are not legitimate tracers of esters in systems involving plasma cholesteryl ester transfer activity. On intravenous injection of doubly labeled high density lipoproteins into rabbits, cholesteryl ester tracer was more rapidly transferred to other lipoprotein fractions than was cholesteryl ether tracer. In direct assays in vitro, the rate of transfer of esters was about two times that of the ether. This difference was not due to tracer impurity or lability of 3H, did not depend on the nature of the donor or acceptor lipoprotein, and was similar for cholesteryl ester transfer activities of both human and rabbit origin.

Animals↗
PubMed1987

The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription.

Using cultures synchronized by elutriator size selection or a feed-starve protocol, we have shown that the CDC8 gene is periodically expressed in the Saccharomyces cerevisiae cell cycle. The transcript level increases some 30-fold in late G1, reaching a peak at approximately the G1/S phase boundary. The timing of this event was compared with those of CDC9 and CDC21, which are already known to be periodically transcribed, and all three genes were found to be expressed at the same time in the cell cycle. In contrast, the histone H2A gene appeared to be expressed distinctly later in the cell cycle than these three genes and this was further investigated by examining expression of all four genes in a cdc4 mutant, held at the restrictive temperature. CDC8, CDC9, and CDC21 were once again expressed together and a complete fluctuation in levels occurred, whereas the histone gene was not expressed, presumably because the cdc4 block point precedes the point of histone expression. The three CDC genes may therefore be coordinately controlled, while the histone gene is regulated separately.

Cell Cycle↗