Elongation Kinetics Research Articles

Altered Flagellar Size‐Control in shf‐1 Short‐Flagella Mutants of Chlamydomonas reinhardtii

ABSTRACTTwo allelic Mendelian mutations which confer a short flagella phenotype were used to explore flagellar size control in Chlamydomonas reinhardtii. When mutant/wild type quadriflagellate dikaryon cells were constructed, their two short flagella rapidly grew out to near wild type length. The kinetics of elongation suggest that the flagellar assembly process is not intrinsically self‐limiting as a number of otherwise attractive models for size control require. Instead, we suggest that there exists a cellular machinery dedicated to flagellar size control and that the short‐flagella mutations alter the machinery in some as yet unknown way. One of the mutants shows temperature‐sensitive flagellar assembly, and both are flagellaless in acetate media. Genetic analysis indicates that the temperaturesensitive, acetate‐sensitive, and short‐flagella phenotypes have a common genetic basis. The responsible gene has been named shf‐1, and it has been mapped to chromosome VI, approximately 5 map units from the centromere.

Complex RNA chain elongation kinetics by wheat germ RNA polymerase II

Kinetics of RNA chain elongation catalyzed by wheat germ RNA polymerase II have been studied using various synthetic DNA templates in the presence of excess dinucleotide monophosphate primers. With single- or double-stranded homopolymer templates, the double reciprocal plots 1/(velocity) as a function of 1/(nucleotide substrate) exhibit positive, negative or no curvature. With poly(dAT) as template, the mechanism of nucleoside monophosphate incorporation into RNA is not the ping-pong kinetic mechanism which was derived for E. coli RNA polymerase (6). Noncomplementary nucleoside triphosphates inhibit RNA transcription allosterically. Cordycepin triphosphate behaves as ATP, and not only inhibits AMP incorporation but also that of UMP and GMP on appropriate templates. The reason for this complex kinetic behavior is not yet understood. Possibilities are raised that there are several nucleoside triphosphate binding sites on wheat germ RNA polymerase II, that additional nucleoside triphosphate dependent enzymatic activities are required for reaction to occur or that the Km value for incorporation of a given nucleoside monophosphate into RNA is dependent on the length of the RNA chain and/or the nucleotide sequence surrounding the complementary base on the DNA template.

Rho-dependent termination of transcription. II. Kinetics of mRNA elongation during transcription from the bacteriophage lambda PR promoter.

The kinetics of elongation of the mRNA that initiates from the lambda PR promoter has been examined using specific restriction fragments as template, and the locations at which significant pausing of the RNA polymerase occurs during in vitro transcription have been established. Major pausing of polymerase, in the absence of rho, occurs at the five rho-dependent termination sites (located between 290 and 450 base pairs downstream from PR) that are described in the accompanying article (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553-9564). The replacement of guanosine by inosine triphosphate in the transcription mix results in the appearance of new pausing sites; these pausing sites correspond, in part, to the new rho-dependent termini between 100 and 260 base pairs from PR identified in the preceding article (cited above) when inosine replaces guanosine in the transcript. The effects of variations in nucleoside triphosphate and salt concentrations on pausing have also been determined. Analysis of the base pair sequences of pausing sites shows that pausing may result from the presence of dyad symmetry, GC-rich sequences, or (for inosine-substituted transcripts) C-rich sequences in the RNA-DNA hybrid region. Quantitation of RNA polymerase pausing at termination loci indicates that pausing sites with relaxation times of 10 to 25 s (at 37 degrees C and 100 to 200 mM KCl) can lead to significant rho-dependent termination. In addition, increasing the length of "natural" pauses by lowering the concentrations of specific nucleoside triphosphate substrates can lead to increased termination efficiency, but only at sites that correspond to rho-dependent termini in elongation experiments conducted at standard concentrations of nucleoside triphosphates. These results, and the findings of the article cited above, are interpreted in terms of a two-component model for rho-dependent termination. Required are: (i) a significant pause in transcript elongation due to sequence and/or structural features at the termination site(s); and (ii) a rho-binding site(s) on the nascent mRNA that is long (70-90 nucleotide residues) and relatively free of secondary structure, and that contains appropriate sequences of cytidine residues.

Head-to-tail polymerization of microtubules in vitro

The kinetics of microtubule polymerization to steady-state and the ability of tubulin subunits to exchange with polymer at steady-state were examined to determine the applicability of the head-to-tail polymerization mechanism (Wegner, 1976) to microtubule assembly in vitro. Under conditions where self-nucleation was a rare event, tubulin was induced to polymerize by the addition of short microtubule fragments, and the kinetics of elongation were analyzed as a pseudofirst-order reaction. At steady-state, a trace amount of [ 3H]tubulin, prepared by labeling in vivo of chick brain protein, was added to polymerized microtubules and the kinetics of label uptake into polymer were monitored by a rapid centrifugal assay. The isotope exchange kinetics were analyzed according to a theoretical model previously applied to actin polymerization (Wegner, 1976) and extended for the case of microtubule polymerization. The rate of head-to-tail polymerization, expressed as the steady-state subunit flux, was 27·6 ± 7·6 per second at 37 °C. The head-to-tail parameter s, a measure of the efficiency of subunit flux, was 0·26 ± 0·07, indicating that four association and four dissociation events resulted in the flux of one subunit through the polymer at steady-state. The role of GTP in this mechanism of microtubule polymerization was examined by replacement of the nucleotide occupying the exchangeable binding site of tubulin with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-methylene)triphosphate. It was found that the rate of steady-state flux was reduced by two orders of magnitude compared to tubulin polymerized with GTP. The head-to-tail parameter approached its limiting value of zero, indicating greatly reduced efficiency of subunit flux through the polymer in the presence of this analog. In summary, this study demonstrates that microtubules exhibit significant headto-tail polymerization in the presence of GTP and, in keeping with theoretical considerations, provides evidence that nucleotide hydrolysis is required for subunit flux through the polymer.

Assembly of tobacco mosaic virus: elongation towards the 3′-hydroxyl terminus of the RNA

The assembly of Tobacco Mosaic Virus (TMV) from its isolated coat protein and RNA is initiated at a single site on the RNA [l] located -1000 nucleotides from the 3’-hydroxyl terminus [2]. A consequence of this internal nucleation site is that subsequent elongation must be bidirectional and while the kinetics and mechanism of growth in the major (3’ to 5’) direction have been extensively studied [3], little is known about growth in the reverse (minor) direction except that it occurs at a considerably slower rate [4-71. We have taken advantage of the possibility of isolating fragments of TMV RNA containing the 3’ terminal region to investigate the kinetics of elongation in the 5’ to 3’ direction. Stripping of TMV under mildly alkaline conditions removes protein specifically from the 5’ end of the particle. Thus nuclease-treated Partially Stripped Virus (PSV) contains fragments of the viral RNA with intact 3’-termini but shortened .5’-tails [8]. RNA molecules with very short tails to the 5’ side of the nucleation region show much less rapid elongation than intact molecules [9] but the reaction does still occur [lo]. Taking advantage of this we have examined the kinetics of elongation towards the 3’ terminus using RNA isolated from nuclease-treated PSV rods. The results indicate that assembly in this direction is more rapid when the protein is supplied in the form of small aggregates (A-protein) than when a disk preparation is used.

Mechanism of Chain Length Determination in Biosynthesis of Milk Fatty Acids

Fatty acid synthetases isolated from all mammalian tissues synthesize predominantly palmitic acid. However, in vivo the mammary gland fatty acid synthetases of some species are responsible for the synthesis of medium chain fatty acids. The objective of this presentation is to outline the mechanism which regulates the product specificity of fatty acid synthetases in general and to illustrate how this control is modified in the mammary gland. Fatty acid synthetases isolated from mammalian tissues are composed of two similar, probably identical, polypeptides, each carrying as many as seven enzyme components. Thioesterase I, the component which functions to terminate growth of acyl chains on the multienzyme, is located at one terminus of each polyfunctional polypeptide and can be detached by limited proteolysis with trypsin. By studying separately the kinetics of chain elongation by the core of the trypsinized complex and of chain termination by the isolated thioesterase I component, it has been possible to establish that the specificities of the elongation and termination reactions account for the synthesis of predominantly the carbon-16 fatty acid by purified fatty acid synthetases. Mammary glands of some species contain an additional enzyme, thioesterase II, which can modify the product specificity of fatty acid synthetase by hydrolyzing medium chain acyl moieties from thioester linkage to the 4′phosphopantetheine of the multienzyme. At all stages of development of rat mammary gland, the amount of theoesterase II present correlates well with the proportion of medium chain fatty acids synthesized by the gland. This mammary gland-specific thioesterase appears responsible for the ability of this tissue to synthesize medium chain fatty acids characteristic of milk fat.

Restriction fragment analysis of bacteriophage SPP1 in vitro transcription by host RNA polymerase.

In vitro transcription of SPP1 DNA occurred on only one of the two strands, the same which is predominantly transcribed in SPP1-infected cells. Transcripts were distributed in several size classes. Analysis of elongation kinetics and of size distribution, coupled with hybridization to DNA restriction fragments, showed that some regions of the template have more initiation sites than others; some have none. Some regions were transcribed directly, some were transcribed from initiation sites located in other regions, and one was never transcribed. Several transcription initiation sites on SPP1 DNA are located on EcoRI fragment 1; four to five others are distributed among other fragments. Cutting the DNA with EcoRI did not introduce artifactual initiation sites. In vitro transcription units can be localized and oriented with respect to the EcoRI restriction map of SPP1 DNA.

Androgenic regulation of elongation of polyribonucleotide chains on rat ventral-prostate chromatin.

The kinetics of polyribonucleotide-chain elongation by rat ventral-prostate RNA polymerase B with homologous chromatin as a template were investigated. Chain elongation was measured under conditions wherein all initiation had occurred, no reinitiation took place and the reaction rate was constant. The kinetic behaviour of prostate RNA polymerase B was consistent with a mathematical model formulated for the multisubstrate enzyme. The addition of each nucleoside triphosphate was independent of the other three. The overall rate of chain elongation was lower when prostate chromatin from castrated rats was used than with prostate chromatin from normal rats. The inclusion of dihydrotestosterone-receptor complexes stimulated the rate of elongation. Androgenic effects did not appear to be directed towards the addition of individual nucleoside triphosphates, but probably towards one of the other major events in RNA-chain elongation, i.e., unwinding of DNA or movement of the enzyme along the template.

Transcriptional control of the isoleucine-valine messenger RNA's in E. coli K-12.

Hybridization of messenger ribonucleic acid (mRNA) isolated from Escherichia Coli K-12 to deoxyribonucleic acid (DNA) from lambdaCI857st68h80dilv was used to detect isoleucine-valine (ilv) specific mRNA. A number of strains partially constitutive for the isoleucine-valine enzymes had levels of ilv mRNA 2 to 3-fold higher than the parent strain. Starvation for any of the branched-chain amino acids resulted in a 20 to 23-fold increase in ilv mRNA as compared to repressed levels. These differences were not due to altered growth rates or to changes in the stability of ilv mRNA. These data indicate that regulation of the isoleucine-valine enzymes by multivalent repression occurs mainly at the level of transcription. Kinetics of elongation of ilv mRNA after repression are consistent with the assumption that the mechanism of multivalent repression involves the prevention of further initiations by RNA polymerase.

Hormonal Regulation of Hypocotyl Elongation inLactuca sativa L.

The kinetics of extension induced by GA31 in the hypocotyl of intact seedlings of Lactuca sativa are similar in the dark and in the light, and differs fundamentally from the kinetics of elongation in the dark without GA3. Both in continuous light and in the dark, GA3-induced promotion starts 24 h after incubation. In the dark, even low concentrations of GA3, which do not affect the length measured after 6 d when the extension of hypocotyl almost ceases, remove the lag period of 48 h which precedes extension, and prolong the high rate of elongation. Following GA3 supply the hypocotyl length in the dark and in the light does not differ until 48 h; thereafter the rate of elongation in the light is less, so that the final length of the hypocotyl is 40 per cent shorter than that of the dark-grown seedlings without GA3. IAA supplied apically to light-grown seedlings induces a weak promotion at a concentration of 1 mg l−1 only. With an increasing concentration of GA3 supplied simultaneously, the concentration of IAA inducing a significant promotion decreases. A combined supply of both these regulators, however, does not restore the light-mediated inhibition of hypocotyl elongation completely. The maximum decrease in hypocotyl length induced by the growth retardants AMO-1618, CCC, and B-9 supplied from the beginning in the dark does not exceed 70 per cent. Saturating doses of GA3 supplied in combination with any one of the retardants compensate only a fraction of the decrease. The results have been interpreted to show that native GAs are not involved in extension growth in the dark.

A semi-quantitative treatment of missense and nonsense suppression in the strA and ram ribosomal mutants of Escherichia coli Evaluation of some molecular parameters of translation in vivo

The efficiency of missense and nonsense suppressors is affected in different ways in Escherichia coli strains carrying different kinds of ribosomal mutations: see Gorini's review (1970). This led Gorini (1971) to postulate a “ribosomal screen” able to distinguish between normal and mutant tRNAs. Here we examine the alternative hypothesis, that the results on suppression can be accounted for by minor changes in the kinetics of polypeptide chain elongation in mutant ribosomes. A general kinetic scheme for the attachment of tRNA to the A site of ribosomes is described. It is postulated that the tRNA first makes a “loose” bond with the codon. A second event is required to stabilize binding and to allow transpeptidation. The probability that the second event occurs is related to the time that the tRNA sticks to the codon in the loose binding state. Ribosomal mutations would make the transition from loose to tight binding more probable ( ram) or less probable ( strA), per collision. A method of analysis is developed that enables one to relate directly the experimental measurements on suppression to molecular parameters. By numerical fitting, it is shown that the following set of conditions can account for the behaviour of the various ribosomal mutants. 1. (a) In the wild-type cell, when a codon becomes associated with its cognate tRNA or release factor, the probability of ensuing chain elongation or termination is very close to unity. The probability of elongation decreases to about one-half in strA1 strains. 2. (b) Loaded nonsense suppressor tRNAs su1, su2 and su3 are present in such amounts that they collide with UAG about as often as the release factor does, but their binding is such that peptide bond formation is not very efficient. 3. (c) In contrast, the low efficiency of two missense suppressors appears related to a relatively low frequency of codon-loaded suppressor collisions, while the association itself may be either stronger ( su58) or much weaker ( su78) than that of the corresponding normal codon-tRNA association.

Flagellum Growth and Regeneration in the True Slime Mould Didymium nigripes

SUMMARY: The kinetics of flagellum elongation during flagellum morphogenesis were investigated in the slime mould, Didymium nigripes. Growth rate varied at different temperatures. Long and short flagella elongated at independent rates and exhibited their maximum rates of elongation at different times. Limitation on final length did not seem to be a function of the precursors available for flagellum synthesis. Growth kinetics of normally growing and regenerating flagella and of amoebae previously exposed to cycloheximide suggest that control of growth rate is not merely a function of flagellum length or the diffusion of precursors to the assembly site. The effect of cycloheximide and streptomycin on flagellum growth were compared in both morphogenetic and regenerating systems. Addition of cycloheximide to a growing or regenerating system immediately halted flagellum elongation. Amoebae exposed to cycloheximide, washed free of the drug and then incubated under standard conditions showed abnormal growth kinetics and reduced final flagellum length; these observations suggest some residual effect of this drug. Streptomycin delayed the morphogenetic process but did not affect flagellum length once morphogenesis occurred.

The dosage-response curve for auxin-induced cell elongation: A reevaluation

The dosage-response curve for auxin-induced growth of coleoptile sections has been reevaluated, using initial growth rates than rates obtained hours after application of auxin. The curve is sigmoid rather than the "classical" bell-shape; i.e., the growth rate increases as the IAA concentration is raised from 10(-8) M, but then the same optimal rate prevails over the range of 3×10(-7) to 10(-3) M. In the absence of sucrose this sigmoid shape persists, but with sucrose present the shape slowly changes into a modified bell-shaped curve. It is suggested that the classical bell-shaped curve is due, at least in part, to an auxin-sucrose interaction, and that it is not a true reflection of the kinetics of auxin-induced elongation.

Studies on the kinetics of ribonucleic acid chain initiation and elongation.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStudies on the kinetics of ribonucleic acid chain initiation and elongationKathleen M. Downey and Antero G. SoCite this: Biochemistry 1970, 9, 12, 2520–2525Publication Date (Print):June 9, 1970Publication History Published online1 May 2002Published inissue 9 June 1970https://doi.org/10.1021/bi00814a019RIGHTS & PERMISSIONSArticle Views44Altmetric-Citations68LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (608 KB) Get e-Alerts Get e-Alerts

Flagellar Development in the Protozoan Peranema trichophorum,

AbstractChanges in flagellar length during the developmental events of flagellar replication and flagellar regeneration were investigated in single cells of the colorless euglenid flagellate Peranema trichophorum at known stages in the life cycle. The leading flagellum could be cut off at any desired length with a tungsten knife operated by a micromanipulator, and the regeneration process recorded photographically.In the regeneration of a flagellum by a postdivision cell, there is a short lag period followed by an elongation with a deceleratory rate. The relationship between elongation rate and flagellar length holds true regardless of the initial stump length. The elongation rates at given lengths are greater in predivision cells than in postdivision cells.At cell division, the parental leading flagellum rapidly shortens at a constant rate and disappears, while simultaneously two new leading flagella elongate at a deceleratory rate. Elongation continues at slower rates throughout interphase.The elongation kinetics of the flagellum during the normal cell cycle, and the kinetics of regeneration at different cell ages have been satisfactorily accounted for by a mathematical model.Shortening and elongation were compared and the relationship between them was discussed in light of the finding that shortening interrupted regeneration in some predivision cells.It was concluded that at least part of the control exerted over flagellar length changes resides in the flagellum itself.The lag period remained the same under a variety of experimental conditions, suggesting that it is a “wound healing response”.