Biomedical subjects

M E Gottesman

Publications and source records attributed to M E Gottesman.

At least 19 recordsLinked to original sources

PubMed1992

Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli.

Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp). Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation. If expressed together, the four hsp are effective at physiological concentrations. Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins.

Bacterial Proteins↗

PubMed1992

The Escherichia coli rpoB60 mutation blocks antitermination by coliphage HK022 Q-function.

The lambdoid bacteriophage regulate gene expression by suppressing transcription terminators. Although similar in sequence to lambda, HK022 lacks an analogue to the lambda N antitermination gene and a distinct nutR sequence. To define the HK022 antitermination system, we plated the phage on Escherichia coli nus mutants that inhibit lambda N function. Only rpoB60 (also called nusC60) blocked HK022 lytic growth. Analyses of HK022-lambda hybrid phage suggested that a HK022 function analogous to lambda Q was inhibited by rpoB60. This result was confirmed with pR'-tR'-galK fusions. HK022 Q-protein suppressed tR' in wild-type but not in rpoB60 mutants. The lambda Q-protein, although inhibited by rpoB60, was more active than the HK022 analogue. A single amino acid difference between the two Q-proteins accounts for the phenotype. Changing the penultimate residue of HK022 Q from alanine to the lambda threonine generated a phage that could propagate on rpoB60 hosts. Host and phage mutations that permitted HK022 growth in rpoB60 strains were characterized. The bacterial suppressors were located in the Escherichia coli nusB gene. The phage suppressors represented recessive mutations in a HK022 b-region sequence encoding an open reading frame of 73 codons.

Amino Acid Sequence↗

PubMed1992

Translation of the prophage lambda cl transcript.

Mutations in rpsB that reduce the levels of the ribosomal protein S2 enhance the translation of cl in lambda lysogens. Two features of the cl transcript are required for enhanced translation: the absence of a leader and the presence of a downstream box, a sequence within the cl coding region that is complementary to the 16S rRNA. 30S ribosomal subunits deficient in S2 form ternary complexes with the cl transcript more efficiently than wild-type subunits. The absence of S2 may change the structure of the 16S rRNA, improving contacts with the cl downstream box.

Bacteriophage lambda↗

PubMed1992

Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Bacterial Proteins↗

PubMed1992

Requirement for E. coli NusG protein in factor-dependent transcription termination.

The 21 kd NusG protein is essential for E. coli viability. Cells depleted for NusG were defective for factor-dependent transcription termination. Rho-induced polarity in the gal operon and the Rho-dependent lambda tR1 and lambda tL1 terminators were suppressed in NusG-deficient cells. NusG depletion inactivated the phage HK022 Nun termination factor. In contrast, the factor-independent lambda tl terminator was fully active in NusG-depleted cells and could be suppressed by phage lambda N function.

Bacterial Proteins↗

PubMed1992

v-ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A.

Ras proteins are membrane-associated transducers of eternal stimuli to unknown intracellular targets. The constitutively activated v-ras oncogene induces dedifferentiation in thyroid cells. v-Ras appears to act by stimulating protein kinase C (PKC), which inhibits the nuclear migration of the catalytic subunit of the cAMP-dependent protein kinase A (PKA). Nuclear tissue-specific and housekeeping trans-acting factors that are dependent on phosphorylation by PKA are thus inactivated. Exclusion of the PKA subunit from the nucleus could represent a general mechanism for the pleiotropic effects of Ras and PKC on cellular growth and differentiation.

Animals↗

PubMed1992

Effect of Escherichia coli nusG function on lambda N-mediated transcription antitermination.

The Escherichia coli Nus factors act in conjunction with the bacteriophage lambda N protein to suppress transcription termination on the lambda chromosome. NusA binds both N and RNA polymerase and may also interact with other Nus factors. To search for additional components of the N antitermination system, we isolated host revertants that restored N activity in nusA1 mutants. One revertant, nusG4, was mapped to the rif region of the E. coli chromosome and shown to represent a point mutation near the 3' end of the nusG gene. The nusG4 mutation also suppressed nusE71 but not nusASal, nusB5, nusC60 (rpoB60), or nusD026 (rho026). However, nusG+ expressed from a multicopy plasmid suppressed nusD026 and related rho mutants for both lambda and phage T4 growth. These results suggest that NusG may act as a component of the N antitermination complex. In addition, the data imply a role for NusG in Rho-dependent termination.

Bacterial Proteins↗

PubMed1992

Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells.

TSH receptor mRNA levels in FRTL-5 thyroid cells are autoregulated at a transcriptional level by the same hormones required for the growth and function of the cells: TSH, insulin, and insulin-like growth factor-I (IGF-I). Thus, the ability of TSH, via its cAMP signal, to down-regulate steady state receptor mRNA levels is preceded by the action of TSH to decrease pre-mRNA levels in nuclear run-on assays to the same quantitative level as evident in Northern analyses. In contrast, the receptor mRNA half-life is shown not to change when down-regulation is reversed by withdrawing TSH in the presence or absence of actinomycin-D. Evidence is additionally provided that TSH receptor mRNA levels are increased by insulin, IGF-I, or calf serum in both Northern and run-on assays. This action cannot be duplicated by hydrocortisone and is evident at more than 20-fold lower concentrations of IGF-I than insulin. Moreover, insulin, IGF-I, and/or calf serum are required for the autoregulatory negative transcriptional regulation of the TSH receptor by TSH/cAMP, as is the case for thyroglobulin. This occurs despite the opposite actions of TSH/cAMP on the two genes, positive in the case of thyroglobulin and negative with TSH receptor. The positive and negative regulatory actions, respectively, of insulin/IGF-I and TSH on receptor gene expression are associated with coincident increases or decreases in cell surface receptors measured by [125I]TSH binding. The autoregulation additionally involves the interplay of a second cAMP-modulated regulatory factor, one which up-regulates TSH receptor mRNA levels rather than causing down-regulation. Thus, cycloheximide inhibits the transcriptional action of both TSH/cAMP and insulin/IGF-I/serum within 4 h, i.e. a rapidly synthesized protein is an intermediate in both cases. The presence of cycloheximide for as little as 1 h, however, uncovers the ability of TSH/cAMP to increase TSH receptor mRNA levels. This activity is the result of the action of a stable cAMP-induced activator which can be detected physiologically, i.e. in the absence of cycloheximide. For example, low levels of a cAMP analog (0.2 mM), as opposed to high levels (greater than 1 mM), can increase TSH receptor RNA levels. Low levels also accelerate the insulin/IGF-I-dependent return of receptor mRNA to normal levels after TSH withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

An Escherichia coli rpoB mutation that inhibits transcription of catabolite-sensitive operons.

The Escherichia coli rpoB636 mutant is defective in the transcription of lac and other catabolite-sensitive operons. The lac promoter variant, UV5, which is independent of cyclic AMP and the cyclic AMP receptor protein, CRP, was also defective in rpoB636 mutants. The activity of the lac UV5 promoter was restored to wild-type levels by deletion of cya (adenylate cyclase) or crp. Cyclic AMP and CRP apparently act as inhibitors of the rpoB636 RNA polymerase.

Bacteriophage lambda↗

PubMed1991

Escherichia coli mutations that block transcription termination by phage HK022 Nun protein.

The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage lambda nut sites. Termination by Nun and antitermination by lambda N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at lambda nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase beta' subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by lambda N function. The mutations antagonized Nun only at lambda nutR; termination at lambda nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the beta' subunit of RNP may be Nun-specific.

Bacteriophage lambda↗

PubMed1991

Reversible inhibition of a thyroid-specific trans-acting factor by Ras.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

Animals↗

PubMed1991

A replica filter assay for expression of ion transport proteins.

In thyroid cells, iodide is accumulated intracellularly via a Na+-I-cotransporter. In this report we show that it is possible to detect diffusible 125I-concentrated in thyroid cell colonies that have been replicated onto nylon filters. Using the replica filter assay, we demonstrate that the iodide transport 1) is restricted to thyroid cells, 2) is Na+ dependent and electrogenic, 3) is inhibited by ClO4- and SCN-, and 4) is adenosine 3',5'-cyclic monophosphate dependent. These are all characteristics of thyroidal iodide transport. This technique can, in principle, detect the expression of any transport system that results in the intracellular accumulation of a diffusible molecule. Moreover, the filter assay can be used to screen for colonies carrying structural or functional mutations affecting such transport systems.

Animals↗

PubMed1990

Renaturation of denatured lambda repressor requires heat shock proteins.

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.

Bacteriophage lambda↗

PubMed1990

Lambda nutR mutations convert HK022 Nun protein from a transcription termination factor to a suppressor of termination.

The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

Bacteriophage lambda↗

PubMed1990

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli.

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.

Alleles↗

PubMed1990

Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon.

Two genes, secE and nusG, situated between the tufB and ribosomal protein rplKAJL operons in the rif region at 90 min on the Escherichia coli chromosome, have been sequenced and characterized. The secE gene encodes a 127-amino-acid-long polypeptide, which is an integral membrane protein essential for protein export (P. J. Schatz, P. D. Riggs, A. Jacq, M. J. Fath, and J. Beckwith, Genes Dev. 3:1035-1044, 1989). The nusG gene encodes a 181-amino-acid-long polypeptide and is involved in transcription antitermination. The protein product of nusG is essential for bacterial viability. The secE-nusG genes are cotranscribed, with transcripts initiated at the PEG promoter and terminated at the Rho-independent terminator in the region of the rplK promoter. The majority of transcripts are processed at a number of sites in the 5' untranslated leader region by RNase III and are possibly also processed by a second unidentified nuclease. The role of transcript processing in the regulation of secE and nusG has not yet been established. The juxtaposition and coregulation of a protein export factor and a transcriptional factor raise questions concerning a functional connection between the two processes.

Amino Acid Sequence↗

PubMed1989

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine.

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.

Animals↗

PubMed1989

Repression of the lambda pcin promoter by integrative host factor.

The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda. The pcin promoter transcribes the cI repressor gene constitutively. lambda cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF). lambda cI- cin-1 grows normally in IHF- mutants, indicating that repressor overproduction from pcin blocks lytic growth. The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription. We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells. In vitro studies show that IHF directly inhibits transcription initiation at pcin. Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter. RNA polymerase bound in an open promoter complex is resistant to IHF. We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure.

Bacterial Proteins↗