Elongation Of Nascent Chains Research Articles

Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation

Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E. coli during the process of export. It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded. Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltosebinding protein has reached molecular weight 33,000 (80% of its entire length) and after ribosebinding protein has been fully elongated (molecular weight 29,000). The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains. In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis. This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed.

Transcriptional and post-transcriptional regulation of globin gene accumulation in murine erythroleukemia cells.

The mechanism responsible for the accumulation of newly synthesized alpha- and beta-globin mRNA in the cytoplasm of induced murine erythroleukemia cells was examined by nuclear mRNA nascent chain elongation (run-off transcription). Hexamethylenebisacetimide, a potent inducer of murine erythroleukemia cell differention, induced high levels of both alpha- and beta-globin gene transcription within 48 to 72 h in culture. Butyric acid, a modest inducer of murine erythroleukemia cells, induced a somewhat lower level of globin gene transcription. With both inducers, alpha-globin transcriptional rates exceeded those of beta-globin. Hemin, on the other hand, showed no detectable increase over the basal rate observed in uninduced cells, even at a time (48 h) when newly synthesized globin mRNA was accumulating in the cytoplasm. These results suggest that there are at least two mechanisms responsible for regulating alpha- and beta-globin structural gene expression in induced murine erythroleukemia cells and that the mechanisms involved are inducer dependent. Hexamethylenebisacetimide and butyric acid increase the rate at which globin genes are transcribed, but hemin appears to allow constitutive levels of transcripts to accumulate.

Transcriptional and post-transcriptional regulation of globin gene accumulation in murine erythroleukemia cells.

The mechanism responsible for the accumulation of newly synthesized alpha- and beta-globin mRNA in the cytoplasm of induced murine erythroleukemia cells was examined by nuclear mRNA nascent chain elongation (run-off transcription). Hexamethylenebisacetimide, a potent inducer of murine erythroleukemia cell differention, induced high levels of both alpha- and beta-globin gene transcription within 48 to 72 h in culture. Butyric acid, a modest inducer of murine erythroleukemia cells, induced a somewhat lower level of globin gene transcription. With both inducers, alpha-globin transcriptional rates exceeded those of beta-globin. Hemin, on the other hand, showed no detectable increase over the basal rate observed in uninduced cells, even at a time (48 h) when newly synthesized globin mRNA was accumulating in the cytoplasm. These results suggest that there are at least two mechanisms responsible for regulating alpha- and beta-globin structural gene expression in induced murine erythroleukemia cells and that the mechanisms involved are inducer dependent. Hexamethylenebisacetimide and butyric acid increase the rate at which globin genes are transcribed, but hemin appears to allow constitutive levels of transcripts to accumulate.

The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication

Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 μ M) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5′- O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP ( K i = 0.4 m M). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655–659,1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens.

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethane sulphonate (EMS) in Chinese hamster V79 cells and the mutagen sensitive derivative V79/79 was investigated by measurement of five parameters: production of strand breaks in template DNA, incorporation of [3H]TdR, semi-conservative and repair synthesis, molecular weights of pulse labelled DNA after mutagen exposure (nascent synthesis) and molecular weights of DNA pulse labelled and chased after mutagen exposure (elongation and ligation). Equal template strand breakage was evident in both cell lines immediately after MNU and EMS exposure and by 4-5 h after MNU the extent of fragmentation was greater in V79/79 cells. After u.v. irradiation template fragmentation was evident in V79/79 but not in V79 cells, even though V79/79 cells failed to excise cyclobutane dimers and repair synthesis was demonstrable in V79 cells but not in V79/79 cells after exposure to all three mutagens. The rate of incorporation of [3H]TdR during semi-conservative DNA synthesis was inhibited equally in a dose dependent manner after u.v. and MNU exposure; incorporation by V79/79 cells was inhibited to a greater extent than by V79 cells after EMS exposure. Nascent DNA synthesis was suppressed more in V79/79 cells than in V79 cells after u.v. but to similar extents in both cell lines after MNU and EMS treatment. Pulse chase experiments indicated a lower rate of elongation of nascent DNA in V79/79 cells after MNU and u.v. exposure but little difference was detectable after EMS.

Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos ( S. purpuratus)

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗ dt = M·f·n· A t , where dP∗ dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and A t = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis ( dP∗ dt ). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo −1 hr −1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo −1 hr −1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes ( f zygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes ( n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on f zygotic and dP∗ dt , the rate of elongation of nascent histone polypeptide chains ( A t ) during the first 6 hr of development was estimated; A t remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

Studies on the mechanism of DNA polymerase alpha. Nascent chain elongation, steady state kinetics, and the initiation phase of DNA synthesis.

A detailed study of the mechanism of nascent chain elongation and of steady state kinetics of purified mouse DNA polymerase alpha has been conducted. Polymerization was examined using a model replication system of poly(dT) as template, oligo(rA) as primer, and dATP as nucleotide substrate, and the probability of chain termination was determined by measurement of the precise chainlength of the products. Reactions were conducted under conditions where products were not utilized as primer. Product chainlength analysis indicated that alpha-polymerase acted in a processive fashion, elongating the primer by the stepwise addition of up to 20 dAMP residues before dissociating. The probability of termination after each dAMP addition depended upon the chainlength of the product and upon the presence of several agents; spermine, spermidine, putrescine, nalidixic acid, or PPi caused a marked increase in termination after the first dAMP addition, and conversely, mouse helix destabilizing protein-1 caused the enzyme to continue extending the same product chain until 18 to approximately 35 dAMP residues had been added. From these and other data, it is concluded that the kinetic mechanisms of termination after the first dAMP addition and after subsequent dAMP additions are different. With this information on how alpha-polymerase elongates a nascent primer(dA)n molecule, a kinetic model and appropriate steady state rate equations were obtained for analysis of substrate initial velocity data and termination probabilities. The substrate kinetic patterns and PPi product inhibition results were consistent with the ordered Ter Ter mechanism Bi Uni Uni Bi Ping Pong proposed in the model, and the model also permits a rational explanation for the differences in termination probability and for the fact that substrate initial velocity plots were linear even though multiple residues of dATP combined with the enzyme during each catalytic cycle. In addition, the results suggest that a rate-limiting step in the steady state occurs at the transition between initiation and elongation, and that higher levels of template.primer increase the rate of this step. This secondary effect of template.primer is discussed in relation to other polymer-forming enzymes, and various kinetic mechanisms which require the presence of two template.primer-binding sites, effector and catalytic, are discussed for their fit to the experimental data.

Polypeptide synthesis catalysed by components of Pichinde virus disrupted by detergent.

Pichinde virus preparations were investigated for ribosomal components and associated activities. After detergent treatment and fractionation, viral components were assayed for polypeptide synthesis, elongation of nascent polypeptide chains, and mRNA activity. It was demonstrated that these subviral particles could synthesize polypeptides when exogenous mRNA template, aminoacyl--tRNAs, translation factors, GTP and appropriate cations were added. Undisrupted, whole virions could not synthesize polypeptide; disruption and subsequent centrifugation of subviral ribosomes was a prerequisite for the biosynthetic activity described.

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. II. Determination of the specific rates of elongation of serum albumin and vitellogenin nascent chains.

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. II. Determination of the specific rates of elongation of serum albumin and vitellogenin nascent chains.

Decreased initiation of DNA synthesis in a temperature-sensitive mutant of hamster cells.

We have analyzed ongoing DNA replication in ts BN-2, a dna- mutant of BHK-21 cells (Nishimoto et al. '78). At the non-permissive temperature of 39.5 degrees C, inhibition of 3H-thymidine into acid-precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S-phase. The fraction of nuclei incorporating 3H-thymidine is similar to that of wild-type cells through the synchronized S-phase of 8 h. Alkaline sucrose gradient analysis shows that pulse-labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at either the permissive or non-permissive temperature. DNA fiber autoradiograms reveal that, at 39.5 degrees C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild-type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN-2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited.

Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions.

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP(1) and VP(2) in addition to the proteins found associated with the viral minichromosome, i.e., VP(3) and histones H(2)A, H(2)B, H(3), and H(4). Our results suggest that the association of VP(1), VP(2), or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP(1) or VP(2) or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome.

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.

Mode of Inhibition of Globin Synthesis by m7G5′;p and m7G5′ppp

The mode of inhibition of rabbit globin synthesis by m7G5'p and m7G5'ppp ("cap analogs") was studied using the rabbit reticulocyte lysate system. The rate of globin synthesis was measured at various concentrations of both f[35S]Met-tRNAf and the cap analogs. The cap analogs were found to inhibit competitively the incorporation of f[35S]Met into hot trichloroacetic acid-insoluble material. Nascent chains prelabelled with f[35S]Met were released at various concentrations of m7G5'ppp. The release of nascent chains was not inhibited by m7G5'p (Suzuki, H. (1976) FEBS Lett. 72, 309) and m7G5'ppp, and it is therefore concluded that the cap analogs inhibit a step of initiation of globin synthesis. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of an 80S/fMet-tRNAf was inhibited by the cap analogs, but not that of a 40S/fMet-tRNAf complex. These data suggest that a factor which is required for the binding of globin mRNA with 40S/fMet-tRNAf complex forms an inactive complex with the cap analogs, so that the cap analogs inhibit globin synthesis.

Inhibition of elongation steps of protein synthesis at reduced potassium concentrations in reticulocytes and reticulocyte lysate

We treated intact reticulocytes with nystatin to make them permeable to small cations and varied the concentration of K+ in the medium; the ionic strength was kept constant by adjusting Na+. As the intracellular K+ was lowered, the rate of protein synthesis decreased. In a reticulocyte lysate, the dependence of the rate of protein synthesis on K+ concentration (at constant ionic strength) was similar to that seen in nystatin-treated cells. We observed no loss of polysomes at low K+. Even when initiation was blocked with aurintricarboxylic acid, decreased K+ inhibited protein synthesis. Analysis of the kinetics of synthesis showed that lowering the K+ concentration inhibits the rate of elongation of nascent chains, but has little effect on the relative rate of initiation of chains.

Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.

Mechanistic aspects of the transfer of nascent periplasmic proteins across the cytoplasmic membrane in Escherichia coli.

Spheroplasts, prepared according to an efficient procedure which is described, secrete a smaller protein fraction of the total cell content than that secreted by intact cells. However, more proteins are found associated to the cell envelopes derived from these spheroplasts than to those derived from intact cells.This result was confirmed when we measured, by specific immunoprecipitation, the amount of secreted and unsecreted alkaline phosphatase. Whereas at any time, at 30 °C, in intact cells 99.6% of the alkaline phosphatase produced is secreted and found in the periplasm [see A. Torriani (1968) J. Bacteriol. 96, 1200–1207], only 40% was secreted by our spheroplast preparation. We suggest that partial depletion of outer‐membrane‐bound protease (or proteases), involved in processing of precursors of secreted protein, is responsible for the significant defect of secretion of precursors which then remain membrane‐bound. Such an experimental protocol has been carried out at various temperatures and the secretion of alkaline phosphatase was assayed. This secretion was found to slightly vary above 22 °C whereas it dramatically decreased below 22 °C. Practically no phosphatase was secreted at 13 °C. However, the synthesis of alkaline phosphatase was not differentially altered at low temperatures compared to total proteins. The inactive presumed precursor of this enzyme which was then not secreted was found associated to the cell envelope. Similar results have been observed in intact cells above 22°C where only the energy of protein synthesis (ΔE= 79 kJ), which causes the elongation of nascent chains, is used for protein secretion; below 22 °C, in the temperature range of the disorder‐order transition of the lipid paraffin chains associated to membrane, a ΔE of 125 kJ is required for secretion of alkaline phosphatase. A ΔE of 146 kJ was found for this process to occur in the same temperature range in spheroplasts. When oleate was incorporated into the membrane of strain K 1059, an unsaturated fatty acid auxotroph, the same result as with wild‐type strains was observed, but a decrease in secretion at high temperatures was induced by this modification of fatty acid composition of the membrane. When elaidate instead of oleate was incorporated into K1059 membrane, the fall in alkaline phosphatase secretion occurred at 33 °C instead of 22 °C, which indicates that the physical state of the lipid phase is really involved in the secretion process. A hypothetical model for secretion of nascent periplasmic proteins, based on the data reported, is presented.

Evidence for acceleration of the rate of elongation of tyrosine aminotransferase nascent chains by dibutyryl cyclic AMP

Analogs of cyclic AMP elevate the synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5) in cultured hepatoma cells and rat liver at a post-transcriptional level but have no discernible effect on total soluble protein synthesis. In order to determine whether cyclic AMP exerts its effect on a step before or after initiation of the synthesis of this enzyme, we have analyzed the ribosomal transit times for both the aminotransferase and total soluble protein in hepatoma cells incubated in the presence or absence of N(6),O(2)'-dibutyryl cyclic AMP. The time required for one ribosome to translate one subunit of the "average" soluble protein (transit time) was about 2 min in cells incubated with or without the cyclic AMP analog. In contrast, the transit time for tyrosine aminotransferase was found to be reduced from 5-8 min under basal conditions to as low as 45 sec after exposure to dibutyryl cyclic AMP. Although the degree of effect varied from experiment to experiment, the relative rate of aminotransferase nascent chain elongation was found to be proportional to the stimulation of its activity. In contrast, dexamethasone did not alter the rate of aminotransferase elongation even though it elevated enzyme activity between 5- and 10-fold. These data are consistent with the hypothesis that induction of tyrosine aminotransferase with cyclic AMP analogs occurs by stimulation of the rate at which ribosomes translate pre-existing mRNA in contrast to adrenal steroids which act by increasing the level of translatable mRNA coding for this enzyme.

Evidence for a function of the adenovirus DNA-binding protein in initiation of DNA synthesis as well as in elongation of nascent DNA chains

Early in lytic infection of KB cells with adenovirus type 5 (Ad5) a viral-coded single-strand-specific DNA-binding protein with a molecular weight of 72,000 is synthesized. The function of this protein in viral DNA synthesis was studied using two different methods for specific inactivation. First, DNA synthesis was examined in KB cells infected with an early temperature-sensitive mutant, H5ts125, which produces a thermolabile DNA-binding protein. At the nonpermissive temperature (40°) the start of the first and subsequent rounds of DNA replication was inhibited. However, viral DNA synthesis in vitro using a system of isolated nuclei obtained from H5ts125-infected KB cells was not thermosensitive compared to wild type. In this in vitro system, replicating molecules were normally converted to mature DNA at 40° without reinitiation of new replication rounds. These results show that thermal inactivation of the H5ts125 DNA-binding protein destroys its capacity to function in the initiation of new replication rounds but does not impair chain growth. As a second method of inactivation we studied viral DNA synthesis in vitro in the presence of antibody against the purified DNA-binding protein. A 50–60% inhibition of the rate of viral DNA synthesis was observed while cellular DNA synthesis was not affected. Density-labeling experiments showed that the antibody inhibits the duplication of both complementary strands, presumably by a decrease of fork movement in replicating adenovirus DNA. The results suggest that the Ad5 DNA-binding protein functions in initiation of DNA replication as well as in elongation of nascent viral DNA chains.

Rate of ribonucleic acid chain growth in Mycobacterium tuberculosis H37Rv

Two methods were employed to measure the rate of ribonucleic acid (RNA) chain growth in vivo in Mycobacterium tuberculosis H37Rv cultures growing in Sauton medium at 37 degrees C, with a generation time of 10 h. In the first, the bacteria were allowed to assimilate [3H]uracil or [3H]guanine into their RNA for short time periods. The RNA was then extracted and hydrolyzed with alkali, and the radioactivity in the resulting nucleotides and nucleosides was measured. The data obtained by this method allowed the calculation of the individual nucleotide step times during the growth of RNA chains, from which the average rate of RNA chain elongation was estimated to be about 4 nucleotides per s. The second method employed the antibiotic rifampin, which specifically inhibits the initiation of RNA synthesis without interfering with the elongation and completion of nascent RNA chains. Usint this method, the transcription time of the 16S, 23S, and 5S ribosomal RNA genes was estimated to be 7.6 min, which corresponds to a ribosomal RNA chain growth rate of 10 nucleotides per s.

Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris · HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase > (C)DNA polymerase β > (C)DNA polymerase α and nuclear(N) poly(A) polymerase > (N)DNA polymerase > (N)RNA polymerase I > (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3′-formyldiphenylhydrazone, rifamycin SV 3′-formyl-n-octyloxime (AF/013) and rifamycin SV 3′-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3′-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3′-formyldinitrophenylhydrazone (AF/DNFI) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3′-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4- N-benzyldemethylrifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3′-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.