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At least 19 recordsLinked to original sources
PubMed1992

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Base Sequence↗
PubMed1992

Parameters affecting transcription termination by Escherichia coli RNA. II. Construction and analysis of hybrid terminators.

Rho-independent terminators are characterized by two major functional regions, one upstream from the termination site having a sequence capable of forming an RNA hairpin in the nascent transcript, the second extending, from the base of this hairpin, seven to nine nucleotides along the transcript to the actual sites of termination (3'-tail region). This latter region of the transcript is often rich in uridine residues. Both regions are postulated to play central roles in the termination process. We have constructed a series of hybrid rho-independent, transcription terminators in which sequences upstream and downstream from the RNA hairpin for the Escherichia coli trp attenuator (trpatt+) are interchanged with sequences from trpatt mutant (1419) or from the phage T7 early terminator (T7Te). Similar hybrids have been constructed for T7Te, replacing flanking sequences with trpatt regions. The effects of such changes on transcription termination have been tested in vitro with purified E. coli RNA polymerase to determine the intrinsic termination efficiency (%T) of each hybrid terminator. Both the trpatt+ terminator and T7Te are highly efficient rho-independent terminators in vitro. However, replacement of trpatt+ sequences upstream and downstream from the RNA-terminator hairpin with the comparable T7Te sequences reduces %T dramatically, suggesting that the RNA-terminator hairpin does not function independently from its flanking regions. Regions downstream from the actual termination/release site are shown to be of considerable importance in determining %T for terminators bearing the T7Te or trpatt1419 3'-tail region, but have little effect on terminators with the trpatt+ 3'-tail region. For terminators bearing the T7Te or trpatt1419 3'-tail region that are inefficient, efficient termination is restored by elevated concentrations of KCl in the reaction. The results do not fit well with models for termination in which %T is determined by a two-step process in which the terminator-RNA hairpin, and a seven to 12 base-pair DNA-RNA hybrid structure rich in uridine residues, act independently to cause the polymerase to pause, and to release the transcript, respectively. DNA sequences both upstream and downstream from these regions, as well as DNA sequences downstream from the transcript termination site, can significantly affect the termination process. Conversely, terminators lacking a 3'-tail region rich in uridine residues can be highly efficient, but only when joined with appropriate sequence immediately downstream from the termination site. This suggests that the 3'-tail region acts in some manner other than the formation of an unstable DNA-RNA hybrid that facilitates termination.

Base Sequence↗
PubMed1989

Terminator-distal sequences determine the in vitro efficiency of the early terminators of bacteriophages T3 and T7.

Bacteriophages T3 and T7 contain homologous terminators for Escherichia coli RNA polymerase that restrict early phage transcription to the leftmost 20% of the linear phage genomes. These two terminators serve equally well as p-independent terminators in vivo, but their in vitro efficiencies and sensitivity to salt and nucleotide concentrations differ dramatically. Sequence analysis shows that the T7 and T3 terminators differ at only two sites in the region normally accepted as defining terminator function. In order to determine which structural features of these two terminators are responsible for their functional differences, a series of hybrid terminators were constructed in which structural features of the two terminators were systematically interchanged. Transcription of hybrid terminator templates revealed that sequences downstream of the termination release sites are responsible for the differences in efficiency of in vitro termination. These sequences also determine the sensitivity of these terminators to elevated salt concentrations and to alterations of substrate concentrations. Alteration of the sequences in the region between three and seven nucleotides downstream of the final T7Te release site is sufficient to reduce termination efficiency to that of T3Te, and point mutations in this region yield terminators with intermediate efficiency. Hence, the determinants of p-independent terminator efficiency in vitro must include elements of the transcription complex other than the structure of the 3' end of the transcript. The termination differences between T7Te, T3Te, and their hybrid derivatives are overcome in vivo; all of these sites become very efficient. This finding further supports the hypothesis that protein factors or other cellular features enhance the efficiency and specificity of p-independent terminators in vivo.

DNA↗
PubMed1985

Comparison of C-terminal and N-terminal PTH in secondary hyperparathyroidism in renal failure.

In 89 patients with disorders in calcium regulation either C-terminal or N-terminal PTH or both, were elevated. In haemodialysis patients the results of C-terminal and N-terminal PTH measurements showed the same trend in 10 patients; in 30 patients only C-terminal and in four patients only N-terminal PTH was elevated. In patients with renal failure, creatinine clearance less than 30 ml/min, both C-terminal and N-terminal PTH were elevated in five patients, C-terminal PTH in 12 patients and N-terminal PTH in none of the patients studied. Fourteen patients with primary hyperparathyroidism were studied; in ten both C-terminal and N-terminal PTH were elevated, in two only C-terminal PTH and in another two only N-terminal PTH was elevated. The results show that there seems to be a better clinical correlation for hyperparathyroidism, in haemodialysis patients and patients with renal failure, using N-terminal PTH determinations rather than C-terminal PTH determinations, because in haemodialysis patients and patients with renal failure split products of the C-terminal region of PTH give unrealistically high PTH results.

Humans↗
PubMed1991

Medial terminal nucleus terminals in the nucleus of the optic tract contain GABA: an electron microscopical study with immunocytochemical double labeling of GABA and PHA-L.

In this study the medial terminal nucleus (MTN) projection to the nucleus of the optic tract (NOT) was investigated in pigmented rats at the light and electron microscopical levels with a new combination of techniques. MTN terminals were anterogradely labeled with Phaseolus vulgaris-leucoagglutinin (PHA-L). Preembedding immunocytochemistry, followed by gold intensification, was used to visualize PHA-L. Postembedding immunocytochemistry with 15 nm immunogold particles was carried out to demonstrate the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Both PHA-L and GABA labeling can be easily discriminated at the electron microscopical level even when present in the same neuronal profiles. Light microscopically MTN-NOT fibers proved to have several branches with many varicosities. MTN terminals were found concentrated in terminal fields. Electron microscopically, it was shown that MTN boutons display characteristics resembling F-type terminals, i.e., terminals with dark mitochondria, pleomorphic vesicles, and symmetrical synapses. All NOT afferents originating from the MTN contained GABA and made multiple contacts exclusively with GABA negative NOT somata and dendrites. These results indicate the existence of a strong and direct inhibitory input onto GABA negative projection neurons in the NOT. This substantiates earlier physiological and morphological reports. It was further demonstrated that the location and organization of MTN terminals in the neuropil differ from that of the retinal input: MTN terminals are largely separated from retinal terminals. MTN terminal fields contain large amounts of GABA positive F terminals in contrast to retinal terminal areas. MTN terminals take part in irregularly shaped agglomerations of terminals, which contain many F terminals and dendritic processes and are surrounded by a glial sheet. Retinal terminals are found grouped together in small circular arrangements contacting a central dendrite.

Animals↗
PubMed1992

Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators.

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.

Base Sequence↗
PubMed1992

Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho.

Rho-dependent terminators constitute one of two major classes of terminators in Escherichia coli. Termination at these sites requires the concerted action of RNA polymerase and rho protein. We present evidence that the efficiency of termination at these sites is governed by kinetic coupling of the rate of transcription of RNA polymerase and the rate of action of rho protein. Termination experiments in vitro indicate that termination efficiency at a rho-dependent terminator is an inverse function of the rate of elongation of RNA polymerase, and each of the mutant phenotypes can be accounted for by the altered rate of elongation of the mutant RNA polymerase. Experiments in vivo show that fast-moving mutant RNA polymerases are termination deficient, while slow-moving mutant RNA polymerases are termination proficient and can suppress the termination deficiency of a slow-acting mutant rho protein. Because of the close coupling of rho action with RNA polymerase, small changes in the elongation rate of RNA polymerase can have very large effects on termination efficiency, providing the cell with a powerful way to modulate termination at rho-dependent terminators.

DNA-Directed RNA Polymerases↗
PubMed1992

Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition.

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

Animals↗
PubMed1992

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. III. Origins and frequency of occurrence of the terminals.

The cell bodies of the layer II/III pyramidal cells in rat visual cortex receive three morphologically distinct types of axon terminals. These axon terminals all form symmetric synapses and have been termed large, medium-sized, and dense axon terminals. The present study shows that each of these different kinds of axon terminals contains gamma-aminobutyric acid (GABA) which suggests that they are inhibitory. From an analysis of the profiles of 50 cell bodies it is calculated that the average layer II/III pyramidal cell has 65 axosomatic synapses, of which 43 are formed by medium-sized terminals, 10 by large terminals, and 12 by dense terminals. Comparison of these different kinds of axon terminals with labelled axon terminals of known origin suggests that the medium-sized terminals are derived from smooth multipolar cells with unmyelinated axons, and that at least some of the dense terminals originate from bipolar cells that contain vasoactive intestinal polypeptides. The source of the large axon terminals is not known, but it is suggested that they originate from multipolar non-pyramidal cells with myelinated axons. Since the initial axon segments of these same neurons receive GABAergic axon terminals from chandelier cells, at least four different types of neurons provide inhibition to the cell bodies and axons of layer II/III pyramidal cells. This serves as an illustration of the complexity of the neuronal circuits in which pyramidal cells are involved.

Animals↗
PubMed1987

nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites.

We have studied the factors that affect transcription termination in vitro at the tR2 terminator of bacteriophage lambda and at the T1 terminator of the Escherichia coli rrnB operon. Termination efficiency at both of these sites is enhanced by the E. coli nusA protein, giving final efficiencies of termination in vitro comparable to those estimated in vivo. Transcripts terminated in the presence of nusA protein are all released from the RNA polymerase complex, indicating that a complete termination reaction is involved, rather than simply induction of a long pause at the terminator. The termination factor activity of the nusA protein does not depend on the presence of rho protein and is not detectably enhanced by that factor. Thus, the nusA protein appears to play a pleiotropic role in E. coli transcription, serving as an antitermination factor, RNA polymerase subunit and true termination factor for some terminator sites.

Bacterial Proteins↗
PubMed1989

Mutant rho factors with increased transcription termination activities. I. Functional correlations of the primary and secondary polynucleotide binding sites with the efficiency and site-selectivity of rho-dependent termination.

We have characterized rho proteins from mutants of Escherichia coli, rho s-81 and rho s-82, which are hyperactive in termination. The two mutant rho proteins are differentially altered both in termination activities and in RNA interactions. rho s-81 generally elicits enhanced termination on various templates such as phage T7 DNA and a DNA restriction fragment containing the trpE intracistronic rho-dependent terminators, either measured as a whole or examined for individual sites. On the other hand, rho s-82 has strikingly different preferences toward individual termination sites, exhibiting overall termination activities higher or lower than normal, depending on templates. From measurements of the rho ATPase activity with T7 RNA and various homoribopolymers as cofactors, both mutant rho proteins are shown to have broadened RNA base specificities in contrast to the stringent requirement for cytosine observed with the wild-type rho. Functional tests on the two kinds of polynucleotide binding sites known for rho have indicated that rho s-81 is mainly altered in the primary site, whereas rho s-82 is simultaneously affected in the secondary binding site as well as the primary site. Thus, we conclude that the primary and secondary sites contribute distinctly in determining the overall efficiency and site-specificity of termination, respectively. Further analysis of detailed termination points at the trpE and lambda tR1 terminators has revealed that major RNA transcripts generated by the wild-type rho and rho s-81 are notably rich in adenine and poor in cytosine for the 3'-terminal five to ten nucleotides, whereas those preferentially terminated by rho s-82 are conversely richer in cytosine than adenine. This finding suggests that rho may recognize the RNA-DNA hybrid region at the 3' end of a nascent transcript in its secondary binding reaction.

Adenosine Triphosphatases↗
PubMed1987

Tau factor from Escherichia coli mediates accurate and efficient termination of transcription at the bacteriophage T3 early termination site in vitro.

The termination signal that limits transcription through the early region of bacteriophage T3 (T3Te) has been cloned and sequenced. The nucleotide sequence of T3Te is identical with that of T7Te, with the exception of a single G to U substitution in the 3' tail of the terminated transcript, and addition of an AC to the loop in the terminator stem-loop, enlarging the loop to six residues. Previous studies of the properties of T3Te have shown that this site is rho independent and is highly efficient for termination in vivo, but is used poorly in vitro during transcription with purified Escherichia coli RNA polymerase. In contrast, the equivalent site in bacteriophage T7 (T7Te) is an efficient termination signal both in vivo and in vitro. However, T3Te becomes an efficient termination site in vitro in the presence of preparations of tau factor. This factor also alters the sites of RNA chain termination found in vitro at T3Te. Transcripts formed in the presence of tau are several nucleotides shorter than those produced with RNA polymerase alone, and have 3' termini that are almost identical with transcripts found in vivo. These latter results are similar to our earlier findings with T7Te, and suggest that other rho independent terminators may act with transcription termination factors in vivo.

Base Sequence↗
PubMed1990

Ultrastructural morphometric analysis of GABA-immunoreactive terminals in the ventrocaudal periaqueductal grey: analysis of the relationship of GABA terminals and the GABAA receptor to periaqueductal grey-raphe magnus projection neurons.

The periaqueductal grey (PAG) plays an important role in the descending modulation of nociception. Inhibitory influences of GABAergic terminals, located within the periaqueductal grey, are thought to play a role in antinociception by influencing the activity of neurons that project to the nucleus raphe magnus and adjacent reticular nuclei. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of GABA-immunoreactive terminals, and to visualize the neuronal distribution of the GABAA receptor in the ventrolateral periaqueductal grey of the rat. Of particular interest was a quantitative description of the interaction between GABA-immunoreactive axon terminals and periaqueductal grey neurons that were retrogradely-labelled from the nucleus raphe magnus and adjacent medullary reticular nuclei. Most terminals were observed to be immediately apposed only to two or three dendrites, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal grey, 37.5% of all GABA-immunoreactive terminals were adjacent to periaqueductal grey-nucleus raphe magnus and periaqueductal grey-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labelled neurons were formed by 17% of GABA-immunoreactive terminals in the ventrolateral periaqueductal grey. We also noted that 13.2% of the GABA-immunoreactive terminals formed symmetrical synapses with GABA-immunoreactive dendrites in the periaqueductal grey, and occasionally those dendrites were retrogradely labelled. Only 0.8% of the GABA-immunoreactive terminals formed putative symmetrical synapses with other GABA-immunoreactive terminals. Consistent with these findings, GABAA receptor immunoreactivity was only associated with dendrites and perikarya in neurons of the ventrolateral PAG. These results are consistent with an inhibitory role for GABA on PAG neurons, a configuration required by hypothetical models for opoid disinhibitory circuitry within the PAG. In addition, the data further suggest that other kinds of GABAergic connections may be important in descending antinociception, and that a population of GABAergic PAG projection neurons exists that may be inhibitory within nucleus raphe magnus and the adjacent reticular nuclei.

Animals↗
PubMed1992

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

Amino Acid Sequence↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗
PubMed1986

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoquinolines↗
PubMed1987

Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal.

Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y. Aloni and N. Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985). In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes. The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract. As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues. Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal. Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl. These results indicate that, at least in isolated nuclei, attenuation can be regulated. The possible involvement of termination factor(s) in the regulation of attenuation is discussed.

Adenoviridae↗