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Jen-Jacobson, L., and L.A. Jacobson (2008) The role of water and the effects of small ions in site-specific protein-DNA interactions. Pp in Structural Biology of Protein-Nucleic Acid Interactions, Rice, P.A., and C. Correll, Ed. Royal Society of Chemistry Publishing, Cambridge, UK Water and small ions play important or even preeminent roles in determining the specificity and thermodynamic properties of protein-DNA interactions. These influences are exerted not only by water or ionic ligands that are released from the macromolecules upon protein-DNA binding, but also by water molecules, and perhaps small ions, that remain bound to the protein-DNA complexes. The release of water from nonpolar surfaces to bulk solvent (hydrophobic effect) is likely the largest single energetic factor that drives site-specific protein-DNA binding. This bulk solvent-release process cannot itself be a determinant of sequence specificity, yet paradoxically enhances specificity because it does not occur unless sequence-specific interactions bring complementary protein and DNA surfaces into sufficiently intimate apposition to expel water from the interface. If we are to aspire to a quantitative rather than qualitative understanding of the roles of water and small ions in macromolecular processes, we will ultimately be compelled to seek deeper knowledge of fundamental physicochemical issues, such as the detailed structure and dynamics of water surrounding macromolecules, the hydration of small ions, and the dynamic behavior of proteins, DNA and their complexes in solution. Szewczyk, N.J., B.K. Peterson, S.J. Barmada, L.P. Parkinson, and L.A. Jacobson (2007) Opposed growth factor signals control protein degradation in muscles of Caenorhabditis elegans. EMBO J 26:935-943 In addition to contractile function, muscle provides a metabolic buffer by degrading protein in times of organismal need. Protein is also degraded during adaptive muscle remodeling upon exercise, but extreme degradation in diverse clinical conditions can compromise function(s) and threaten life. Here, we show how two independent signals interact to control protein degradation. In striated muscles of Caenorhabditis elegans, reduction of insulin-like signaling via DAF-2 insulin/IGF receptor or its intramuscular effector PtdIns-3-kinase (PI3K) causes unexpected activation of MAP kinase (MAPK), consequent activation of pre-existing systems for protein degradation, and progressive impairment of mobility. Degradation is prevented by mutations that increase signal downstream of PI3K or by disruption of autocrine signal from fibroblast growth factor (FGF) via the FGF receptor and its effectors in the Ras-MAPK pathway. Thus, the activity of constitutive protein degradation systems in normal muscle is minimized by a balance between directly interacting signaling pathways, implying that physiological, pathological, or therapeutic alteration of this balance may contribute to muscle remodeling or wasting.
Szewczyk, N.J., I.A. Udranszky, E. Kozak, J. Sunga, S.K. Kim, L.A. Jacobson, and C.A. Conley (2006) Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. J. Exp. Biol. 209:4129-4139 Studies of the model organism Caenorhabditis elegans have almost exclusively utilized growth on a bacterial diet. Such culturing presents a challenge to automation of experimentation and introduces bacterial metabolism as a secondary concern in drug and environmental toxicology studies. Axenic cultivation of C. elegans can avoid these problems, yet past work suggests that axenic growth is unhealthy for C. elegans. Here we employ a chemically defined liquid medium to culture C. elegans and find development slows, fecundity declines, lifespan increases, lipid and protein stores decrease, and gene expression changes relative to that on a bacterial diet. These changes do not appear to be random pathologies associated with malnutrition, as there are no developmental delays associated with starvation, such as L1 or dauer diapause. Additionally, development and reproductive period are fixed percentages of lifespan regardless of diet, suggesting that these alterations are adaptive. We propose that C. elegans can exist as a healthy animal with at least two distinct adult life histories. One life history maximizes the intrinsic rate of population increase, the other maximizes the efficiency of exploitation of the carrying capacity of the environment. Microarray analysis reveals increased transcript levels of daf-16 and downstream targets and past experiments demonstrate that DAF-16 (FOXO) acting on downstream targets can influence all of the phenotypes we see altered in maintenance medium. Thus, life history alteration in response to diet may be modulated by DAF-16. Our observations introduce a powerful system for automation of experimentation on healthy C. elegans and for systematic analysis of the profound impact of diet on animal physiology.
Szewczyk, N.J., and L.A. Jacobson (2005) Signal-transduction networks and the regulation of muscle protein degradation. Int. J. Biochem. Cell Biol. 37:1997-2011 Protein degradation in muscle functions in maintaining normal physiological homeostasis and adapting to new homeostatic states, and is required for muscle wasting or atrophy in various pathological states. The interplay between protein synthesis and degradation to maintain homeostasis is complex and responds to a variety of autocrine and intercellular signals from neuronal inputs, hormones, cytokines, growth factors and other regulatory molecules. The intracellular events that connect extracellular signals to the molecular control of protein degradation are incompletely understood, but likely involve interacting signal-transduction networks rather than isolated pathways. We review some examples of signal-transduction systems that regulate protein degradation, including effectors of proteolysis inducing factor (PIF), insulin and insulin-like growth factor (IGF) and their receptors, and fibroblast growth factor (FGF) and its receptors.
Szewczyk, N.J., and L.A. Jacobson (2003) Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans. EMBO J. 22:5058-5067 Signaling by fibroblast growth factors (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL-15 FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL-15, by means of a reduction of function mutation (clr-1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre-existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl-17 or let-756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen-activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a growth factor receptor using an identified signal transduction pathway. The data raise the possibility that FGF-triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.
Fostel, J.L., L.A. Coste, and L..A. Jacobson (2003) Degradation of transgene-coded and endogenous proteins in the muscles of Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 312:173-177 To develop reporter systems to study the regulation of protein degradation in innervated muscle, we have used strains of the nematode Caenorhabditis elegans containing transgenes that fuse lacZ or Green Fluorescent Protein (GFP) coding regions to muscle-specific promoter/enhancer regions, such that the fusion proteins are expressed exclusively in body-wall and vulval muscle cells. The starvation-induced degradation of the ß-galactosidase reporter protein is quantitatively similar to that of two endogenous muscle proteins, arginine kinase and adenylate kinase. A soluble GFP in the muscle cytosol is degraded during starvation, but when GFP is fused to a full-length myosin heavy chain and incorporated into myofibrils, it is resistant to starvation-induced degradation. This suggests that under some conditions soluble muscle proteins may be extensively catabolized in preference to the proteins of the contractile fibers. Szewczyk, N.J., B.K. Peterson, and L.A. Jacobson (2002) Activation of Ras and the MAP kinase pathway promotes protein degradation in muscle cells of Caenorhabditis elegans. Mol. Cell. Biol. 22:4181-4188 To discover and study intracellular signals that regulate proteolysis in muscle, we have employed transgenic strains of Caenorhabditis elegans that produce a soluble LacZ reporter protein limited to body-wall and vulval muscles. This reporter protein is stable in well-fed wild-type animals, but its degradation is triggered upon a shift to 25 degrees C in a strain carrying a temperature-sensitive activating mutation in the Ras oncogene homologue let-60. These mutants are not physiologically starved, inasmuch as growth rates are normal at 25 degrees C. Ras-induced degradation is not prevented by the presence of cycloheximide added at or before the temperature shift and thus uses preexisting proteolytic systems and signaling components. Furthermore, degradation is triggered when adult animals are shifted to conditions of 25 degrees C, confirming that Ras acutely promotes protein degradation in muscles whose developmental history is normal. Reduction-of-function mutations in the downstream protein kinase Raf (lin-45), MEK (mek-2), or mitogen-activated protein kinase (MAPK) (mpk-1) prevent Ras-induced protein degradation, whereas activated MPK-1 is sufficient to trigger degradation, indicating that this kinase cascade is the principal route by which Ras signaling triggers protein degradation in muscle. This pathway is activated in hypodermal cells by the LET-23 epidermal growth factor receptor homologue, but an activating mutation in let-23 does not promote proteolysis in muscle. Starvation-induced LacZ reporter degradation is unaffected by reduction-of-function mutations in Ras, Raf, MEK, or MAPK, implying that Ras activation and starvation trigger proteolysis by mechanisms that are at least partially independent. This is the first evidence that Ras-Raf-MEK-MAPK signaling activates protein degradation in differentiated muscle.
Jen-Jacobson, L., L. Engler, and L.A. Jacobson (2000) Structural and thermodynamic strategies for site-specific DNA binding proteins. Structure Fold Des. 8:1015-1023 Background: Site-specific protein-DNA complexes vary greatly in structural properties and in the thermodynamic strategy for achieving an appropriate binding free energy. A better understanding of the structural and energetic engineering principles might lead to rational methods for modification or design of such proteins. Results: A novel analysis of ten site-specific protein-DNA complexes reveals a striking correspondence between the degree of imposed DNA distortion and the thermodynamic parameters of each system. For complexes with relatively undistorted DNA, favorable enthalpy change drives unfavorable entropy change, whereas for complexes with highly distorted DNA, unfavorable DeltaH degrees is driven by favorable DeltaS degrees. We show for the first time that protein-DNA associations have isothermal enthalpy-entropy compensation, distinct from temperature-dependent compensation, so DeltaH degrees and DeltaS degrees do not vary independently. All complexes have favorable DeltaH degrees from direct protein-DNA recognition interactions and favorable DeltaS degrees from water release. Systems that strongly distort the DNA nevertheless have net unfavorable DeltaH degrees as the result of molecular strain, primarily associated with the base pair destacking. These systems have little coupled protein folding and the strained interface suffers less immobilization, so DeltaS degrees is net favorable. By contrast, systems with little DNA distortion have net favorable DeltaH degrees, which must be counterbalanced by net unfavorable DeltaS degrees, derived from loss of vibrational entropy (a result of isothermal enthalpy-entropy compensation) and from coupling between DNA binding and protein folding.Conclusions: Isothermal enthalpy-entropy compensation implies that a structurally optimal, unstrained fit is achieved only at the cost of entropically unfavorable immobilization, whereas an enthalpically weaker, strained interface entails smaller entropic penalties. Szewczyk, N.J., J.J. Hartman, S.J. Barmada, and L.A. Jacobson (2000) Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans. J. Cell Sci. 133:2003-2010 A myosin-lacZ fusion, expressed in 103 muscle cells of Caenorhabditis elegans, reports on how proteolysis in muscle is controlled by neural and intramuscular signals. Upon acute starvation, the fusion protein is degraded in the posterior 63 cells of the body-wall muscle, but remains stable in 32 anterior body-wall muscles and 8 vulval muscle cells. This distinction correlates with differences in the innervation of these cells. Reporter protein in the head and vulval muscles becomes labile upon genetic 'denervation' in mutants that have blocks in pre-synaptic synthesis or release of acetylcholine (ACh) or post-synaptic reception at nicotinic ACh receptors (nAChR), whereas protein in all 103 muscles is stabilized by the nicotinic agonist levamisole in the absence of ACh production. Levamisole does not stabilize muscle protein in nAChR mutants that are behaviorally resistant to levamisole. Neural inputs thus exert negative control over the proteolytic process in muscle by stimulating muscle nicotinic ACh receptors. Full text of this article can be obtained from http://www.biologists.com/JCS/113/11/jcs1231.html.
Zdinak, L., I. Greenberg, N. Szewczyk, S. Barmada, M. Cardamone-Rayner, J. Hartman, and L. Jacobson (1997) Transgene-coded chimeric proteins as reporters of intracellular proteolysis: starvation-induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans. J. Cell. Biochem. 67:143-153 The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in-frame fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer. Ashcom, J.D., and L.A. Jacobson (1989) Self-quenched fluorogenic protein substrates for the detection of cathepsin D and other protease activities. Anal. Biochem. 176:261-264 Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including cathepsin D. Hawdon, J., S. Emmons, and L. Jacobson (1989) Regulation of proteinase levels in the nematode Caenorhabditis elegans: Preferential depression by acute or chronic starvation. Biochem. J. 264:161-165 Acute starvation of the wild-type of the nematode Caenorhabditis elegans depresses the level of cathepsin D by 65% within 4-8 h and the level of the thiol cathepsins Ce1 and Ce2 to about the same extent after 24 h. There is no parallel loss of lysosomal beta-glucosidase or beta-hexosaminidase activities. In strains which are chronically starved as a result of mutations which compromise feeding behaviour (unc-52) or nutrient uptake into the intestinal cells (daf-4), cathepsin D levels are decreased to about 15% of the level in fully fed wild-type animals. We suggest that the decline in the cathepsin D level results from autodigestion when alternative protein substrates are depleted in the lysosomes.
Sarkis, G.J., M.R. Kurpiewski, J.D. Ashcom, L. Jen-Jacobson, and L.A. Jacobson (1988) Proteases of the nematode Caenorhabditis elegans. Arch. Biochem. Biophys. 261:80-90 Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate. Sarkis, G., J. Ashcom, J. Hawdon, and L. Jacobson (1988) Decline in protease activities with age in the nematode Caenohabditis elegans. Mech. Ageing Dev. 45:191-201 The activities of 3 lysosomal proteases in the nematode Caenorhabditis elegans are markedly lower in older animals. The aspartyl protease cathepsin D declines about 10-fold from day 3 (early adulthood) to day 11 (near the mean lifespan); this reflects a net decline in the amount of cathepsin D antigen. The specific activity of the thiol protease cathepsin Ce1 declines about 2.5-fold over the same period, and the specific activity of the thiol protease cathepsin Ce2 declines about 8-fold. The activity of a new non-lysosomal protease, designated cathepsin CeX, is invariant with age. The data are consistent with the hypothesis that reduced protease activity in older animals may cause a decline in the rate of protein turnover with age, but do not prove this hypothesis. Jacobson, L., L. Jen-Jacobson, J. Hawdon, G. Owens, M. Bolanowski, S. Emmons, M. Shah, R. Pollock, and D. Conklin (1988) Identification of a putative structural gene for cathepsin D in Caenorhabditis elegans. Genetics 199:355-363 Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.
Clokey, G.V., and L.A. Jacobson (1986) The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech. Ageing Dev. 35:79-94 The nematode Caenorhabditis elegans contains autofluorescent lipofuscin granules, located exclusively in the 32-34 intestinal cells. Using epifluorescence microscopy on live adult animals, we have shown that fluorescent-labeled exogenous probes are taken up by endocytosis and accumulate within the granules. Macromolecular solutes such as proteins and dextran appear to be taken up by fluid-phase pinocytosis. There is no phagocytosis of latex particles with diameter greater than or equal to 0.25 micron. The granules concentrate the lysosomotropic weak base acridine orange, indicating that they have an acidic internal milieu. These observations imply that the lipofuscin granules in the intestinal cells are secondary lysosomes which remain active recipients of endocytosed materials. Russell, R.L., and L.A. Jacobson (1985) Some aspects of aging are easily studied in nematodes (a review). Pp 128-145 in Handbook of the Biology of Aging, Finch, C.E., and E.L. Schneider, Ed. Van Nostrand-Reinhold, New York Wnek, A.P., and L.A. Jacobson (1985) The size and heterogeneity of the messenger RNA associated with 70 S monosomes from down-shifted Escherichia coli. Arch. Biochem. Biophys. 241:106-117 After an energy source shift-down, Escherichia coli accumulates 70 S ribosome-mRNA complexes ("70 S monosomes"). The monosome mRNA strands are predominantly primary transcription products with purine nucleoside 5'-triphosphate and 5'-diphosphate termini present at a 1:2 ratio. The number-average chain length is 564 +/- 30 nucleotides, indicating that the population represents primarily monocistronic mRNAs. Digestions with endonucleases and exonucleases indicate that the ribosomes lie near the 5' ends of the mRNA strands and that the majority of the mRNA strands contain 5'-proximal "leader" sequences (average 10 nucleotides) outside the protective boundary of the ribosome. These data are consistent with the hypothesis that the increased functional stability of mRNA in down-shifted cells may result from protection by bound ribosomes of endonuclease-susceptible site(s) near the 5' ends of the mRNA strands. Jacobson, L.A., L. Jen-Jacobson, and A.P. Wnek (1985) The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli. Arch. Biochem. Biophys. 241:118-131 The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site. Bolanowski, M.A., L.A. Jacobson, and R.L. Russell (1983) Quantitative measures of aging in the nematode Caenorhabditis elegans: II. Lysosomal hydrolases as markers of senescence. Mech. Ageing Dev. 21:295-319 In an attempt to provide additional quantitative markers of senescence in the nematode Caenorhabditis elegans, we have identified age-dependent increases in four lysosomal enzymes: acid phosphatase, beta-N-acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D-mannosidase. These enzymes were judged to be lysosomal on the basis of their resemblance to analogous mammalian lysosomal enzymes with regard to subcellular fractionation, lectin binding, Km, molecular weights, inhibitor sensitivities, and pH optima. In nematode populations which had a median lifespan of 8.9 +/- 0.7 days and a maximum lifespan of 14-16 days, we observed the following increases in acid hydrolase activities per animal from day 3 (early adulthood) to day 10: (1) up to 2.5-fold for acid phosphatase; (2) 8-fold for beta-N-acetyl-D-glucosaminidase; (3) 9-fold for beta-D-glucosidase; and (4) 4-fold for alpha-D-mannosidase. Three forms of acid phosphatase and two forms of beta-D-glucosidase were separated by ion-exchange chromatography, but in each case only one form of the enzyme was primarily responsible for the age-dependent increase in total activity: acid phosphatase I increased 18-fold, while beta-D-glucosidase I increased 100-fold. By contrast, there were only slight age-dependent changes in choline acetyltransferase, acetylcholinesterase, or alpha-D-glucosidase activities after early adulthood. The age-dependent increases in acid phosphatase, beta-N-acetyl-D-glucosaminidase, beta-D-glucosidase, and alpha-D-mannosidase activities are sufficiently large and reproducible to be useful quantitative markers of senescence in C. elegans. Jacobson, L.A., and L. Jen-Jacobson (1982) Post-transcriptional control of protein synthesis during substrate adaptation. Pp 191-204 in Experiences in Biochemical Perception, Ornston, L.N., and S. Sligar, Ed. Academic Press, New York Bolanowski, M.A., R.L. Russell, and L.A. Jacobson (1981) Quantitative measures of aging in the nematode Caenorhabditis elegans. I. Population and longitudinal studies of two behavioral parameters. Mech. Ageing Dev. 15:279-295 As a first step in the quantitative characterization of senescence in the nematode Caenorhabditis elegans, we have studied movement wave frequency, defecation frequency, and whole-body water efflux as a function of age. Populations of C. elegans, strain N2, were cultured monoxenically on E. coli lawns at 20 degrees C. The median lifespan in such populations was approximately 12 days. Population mean movement wave frequency declined linearly with age (slope = -4.66 waves/minute per day). The decline in population mean defecation frequency (defecations per minute) was multiphasic, consisting of (1) a rapid decline (slope = -0.233 defecations/minute per day) from day 3 to day 6, (2) no apparent trend from day 6 to day 9, and (3) a gradual decline (slope = -0.089 defecations/minute per day) from 9 to day 14. Animals alive on or after day 15 were not observed to defecate. In longitudinal studies, individual animals exhibited linear declines in movement wave frequency and multiphasic declines in defecation frequency. For future population studies, the age-dependent declines in movement and defecation frequency appear sufficiently large and reproducible to a multiparametric description of senescence in C. elegans. One physiological parameter, 3H2O efflux, was found to be age-independent and to consist of two first-order rates. The half-times of the slow and fast efflux rates were approximately 15 and approximately 2.1 minutes, respectively. The two half-times and the fractions of 3H2O exhibiting the two half-times were invariant with age. Jacobson, L.A., and L. Jen-Jacobson (1980) Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down. J. Bacteriol. 142:888-898 We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.
Jacobson, L.A., and L. Jen-Jacobson (1980) Control of protein synthesis in Escherichia coli: lack of correlation with changes in intracellular pools of ATP, GTP, and ppGpp. Arch. Biochem. Biophys. 203:691-696 Leschine, S.B., and L.A. Jacobson (1979) Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down. J. Virol. 30:267-278 Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.
Jacobson, L.A., and J.C. Baldassare (1976) Association of messenger ribonucleic acid with 70S monosomes from down-shifted Escherichia coli. J. Bacteriol. 127:637-643 The complexed 70S ribosomes (monosomes) that accumulate in Escherichia coli after an energy source shift-down were examined in an electron microscope. In all cases, the ribosomes lie at or near one end of a ribonucleic acid (RNA) strand. This messenger RNA (mRNA) has a mean length of 168 nm and a length-average length of 200 nm, sufficient to code for polypeptides of a weight-average molecular weight of 20,000. The length distribution indicates that these strands are a reasonable representation of the population of monocistronic mRNA's of E. coli. The mRNA strands disappear entirely upon digestion with pancreatic ribonuclease, phosphodiesterase I, or polynucleotide phosphorylase. The susceptibility to digestion by 3'-exonucleases indicate that the ribosomes lie at the 5' end of the mRNA strands. These results are consistent with the hypothesis that down-shifted cells have a translational defect at a point subsequent to the binding of ribosomes to mRNA but prior to the formation of the first peptide bond, such that ribosomes remain bound at or near their points of initial attachment to mRNA.
Westover, K.C., and L.A. Jacobson (1974) Control of protein synthesis in Escherichia coli. II. Translation and degradation of lactose operon messenger ribonucleic acid after energy source shift-down. J. Biol. Chem. 249:6280-6287
Westover, K.C., and L.A. Jacobson (1974) Control of protein synthesis of Escherichia coli. I. Translation and functional inactivation of messenger ribonucleic acid after energy source shift-down. J. Biol. Chem. 249:6272-6279
Jacobson, L.A. (1972) Control of stable ribonucleic acid chain initiation in Escherichia coli during diauxie lag. J. Bacteriol. 109:678-685
Ruscetti, F.W., and L.A. Jacobson (1972) Accumulation of 70S monoribosomes in Escherichia coli after energy source shift-down. J. Bacteriol. 111:142-151
Jacobson, L.A. (1971) Evidence for the polycistronic transcription of ribosomal RNA in Escherichia coli. Biochem. Biophys. Res. Commun. 43:372-377 Jacobson, L.A. (1970) Regulation of ribonucleic acid synthesis in Escherichia coli during diauxie lag: accumulation of heterogeneous ribonucleic acid. J. Bacteriol. 102:740-746
Gunsalus, I.C., 2.n.d. Bertland AU, and L.A. Jacobson (1967) Enzyme induction and repression in anabolic and catabolic pathways. Arch. Microbiol. 59:113-122 Jacobson, L.A., R.C. Bartholomaus, and I.C. Gunsalus (1966) Repression of malic enzyme by acetate in Pseudomonas. Biochem. Biophys. Res. Commun. 24:955-960 Gunsalus, I.C., H.E. Conrad, P.W. Trudgill, and L.A. Jacobson (1964) Regulation of catabolic metabolism. Israel J. Med. Sci. 1:1099 Jacobson, L.A., A.U. Bertland, and I.C. Gunsalus (1964) Biochemical relationship of steroids and monoterpenoids in fluorescent pseudomonads. Bact. Proc. 1964:105 |
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