Cyclic AMP receptor protein: role in transcription activation.

The structure of this pleiotropic activator of gene transcription in bacteria and its interaction sites at promoter DNA's as well as the role of this protein in the RNA polymerase-promoter interactions are reviewed.

Identification of a prime suspect.

Recent findings help to define the multiple functions of the sigma subunit of bacterial RNA polymerase, from promoter recognition to the release of pausing during initial RNA elongation; these functions can now be confronted with a crystal structure of an essential domain of the sigma subunit.

The HIV-1 repeated sequence R as a robust hot-spot for copy-choice recombination.

Template switching during reverse transcription is crucial for retroviral replication. While strand transfer on the terminal repeated sequence R is essential to achieve reverse transcription, template switching from internal regions of the genome (copy choice) leads to genetic recombination. We have developed an experimental system to study copy-choice recombination in vitro along the HIV-1 genome. We identify here several genomic regions, including the R sequence, where copy choice occurred at high rates. The frequency of copy choice occurring in a given region of template was strongly influenced by the surrounding sequences, an observation that suggests a pivotal role of the folding of template RNA in the process. The sequence R, instead, constituted an exception to this rule since it was a strong hot-spot for copy choice in the different sequence contexts tested. We suggest therefore that the structure of this region has been optimised during viral evolution to ensure efficient template switching independently from the sequences that might surround it.

Two Escherichia coli fructose-6-phosphate kinases. Preparative purification, oligomeric structure and immunological studies.

Two isoenzymes of fructose-6-phosphate kinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) are present in Escherichia coli K12. One isoenzyme is allosterically inhibited by phosphoenolpyruvate and activated by nucleoside diphosphates, and is a tetramer composed of four subunits of molecular weight 35 000. A simple method for the purification of this enzyme is reported. Equilibrium dialysis indicates that there are four ATP sites and four GDP sites per tetramer. The second isoenzyme is present in low quantity in wild type bacteria. This enzyme is devoid of allosteric properties. A complete method of purification is described. Determination of its molecular weight under native and denaturing conditions indicates that this protein is a dimer composed of two subunits of molecular weight 36 000. Antisera have been produced against both isoenzymes. The antiserum against one isoenzyme does not cross-react with the other. Discrepancies between our results and those of other workers are discussed.

Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli.

Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions. Their kinetic behavior has been characteriized with respect to different effectors and substrates. The conclusions reached on one hand by Malcovati and Kornberg (Biochim. Biophys. Acta (1969) 178, 420-423), on the other hand by Fraenkel, Kotlarz and Buc (J. Biol. Chem. (1973) 248, 4865-4866) have been found to be true in aerobiosis as well as in anaerobiosis. The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions. Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time.

Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes.

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [14C]glucose glycogen labelling (20 min) earlier than with insulin (30-40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2-3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 microM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.

A transcriptionally active plasmid-protein complex isolated from Escherichia coli.

A stable transcriptionally active plasmid-protein complex has been isolated in high yield from Escherichia coli containing the thermally-inducible plasmid pKN 402A. The complexes which have a protein/DNA weight ratio of approx. 1 contain more than 11 polypeptide species. The weight percents of the three known proteins in the complex H1, RNA polymerase and HU, are 23, 23 and 5%, respectively. In vitro RNA synthesis by this complex proceeds for several hours and is inhibited by rifampicin and actinomycin to 33 and 98%, respectively, suggesting that most of the observed nucleotide incorporation is due to elongation of preinitiated RNA chains. Exogenous E. coli RNA polymerase but not exogenous DNA stimulates the in vitro transcription indicating that RNA polymerase is limiting and binds tightly to the plasmid. Stimulation of the in vitro transcription by the addition of exogenous E. coli core polymerase suggests that sigma subunit may be released in the RNA synthesis. This transcriptionally active complex should prove to be useful to study the mechanism of transcription and regulation in vivo.

The metabolic effects of oxalate on intact red blood cells.

The metabolic effects of oxalate on pyruvate kinase were studied in intact human red blood cells and compared to the spontaneous modifications induced by congenital pyruvate kinase deficiency. In normal cells, oxalate (2-3 . 10(-4) M) produces a large increase of the monophosphoglycerates, phosphoenolpyruvate pool and decrease of pyruvate concentrations as a result of pyruvate kinase inhibition; it does not significantly modify 2,3-diphosphoglycerate level, ATP formation or overall glycolytic activity. Those effects of oxalate are not due to Mg2+ chelation. A similar metabolite pattern is observed in vivo in erythrocytes with congenital pyruvate kinase deficiency, in which ATP concentration and glycolytic activity are described. These cells are more sensitive to oxalate than normal ones. Results are discussed with reference to the rate-limiting character of normal or congenitally deficient pyruvate kinase.

The effect of adenosine and chloroadenosine on sex differences in the energy metabolism of rat hepatocytes.

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).

Influence of oxalate on the rate of the tricarboxylic acid cycle in rat hepatocytes.

In hepatocytes isolated from fed rats, physiological concentrations of oxalate lower the flux through the tricarboxylic acid cycle (-48%) and reduce the steady-state levels of oxaloacetate and other Krebs cycle intermediates. All the metabolic modifications observed are explained by pyruvate carboxylase inhibition, since oxalate hardly modifies the flux through pyruvate dehydrogenase.

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli.

Two Escherichia coli control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites P1 and P2, both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter. The final complexes were characterized by their topological unwinding, by DNase I and orthophenanthroline footprints, as well as by methylation of unpaired cytosine residues. At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow. The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of nucleotide substrates as well as during an abortive initiation cycle. At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period. Not all the final species are active and unwinding appears to precede, in time, full open-complex formation. At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity. From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing. Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, P1 and P2, on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones). We conclude that negative supercoiling is not an extra-regulatory element of the lactose system, allowing modulation of expression of the wild-type promoter to the profit of P1. Instead, P2 and P1, in the absence of active catabolite receptor protein (CRP-cAMP), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined. The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned. The major effect of CRP-cAMP stimulation appears to be the direct activation at the P1 promoter.

Compression of the DNA minor groove is responsible for termination of DNA synthesis by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) generally terminates plus strand DNA synthesis at the centre of the viral genome. The central termination sequence (CTS) contains two termination sites which are located at the 3' end of AnTm motifs. These motifs generate a global curvature of the DNA helix which correlates with termination of DNA synthesis. Here, we have characterized HIV-1 RT termination sites on different DNA sequences. Again, they are located at the 3' end of A-tracts. Using hydroxyl radicals as a probe of the width of the DNA helix, we have shown that RT termination sites are always located a few base-pairs downstream of a compressed minor groove. Mutations which relieve these compressions also abolish the termination events. The replacement of the adenine tracts by 2,6-diaminopurine tracts has a similar effect. Moreover, no termination site is observed on DNA sequences containing phased GC-tracts which curve the DNA helix but compress the major groove. The compression of the DNA minor groove and not necessarily the curved trajectory of the DNA is, therefore, responsible for termination of DNA synthesis at the CTS by HIV-1 RT. This conclusion is consistent with interpretation of other biochemical data on the processivity of HIV-1 RT, based on the structure of a DNA-enzyme complex.

Recombination during reverse transcription: an evaluation of the role of the nucleocapsid protein.

The human immuno deficiency virus type 1 nucleocapsid protein 7 (HIV-1 NCp7) is a major component of the reverse transcription complex. Its effect on reverse transcription and homologous recombination has been studied in vitro under strictly identical experimental conditions. For high enzyme concentrations, NCp7 did not stimulate DNA synthesis. The time-course for completion of reverse transcription as well as the processivity and the pattern of pausing were similar in the presence or absence of NCp7. However, the addition of NCp7 significantly affected the yield of the reaction, a decrease exacerbated as the length of the copied RNA increased. We attribute this phenomenon to a destabilization of the RNA/DNA duplex at intermediate stages of reverse transcription.In contrast, NCp7 enhanced homologous recombination during synthesis mediated by HIV-1 RT (reverse transcriptase), as it did for Moloney murine leukemia virus RT. On naked RNA the process of recombination was dependent on the concentration of RT, suggesting that binding of RT to an intermediate of strand transfer was the limiting step. This dependence was relieved in the presence of NCp7. This effect does not imply a direct interaction between RT and NCp7, since similar results were obtained when NCp7 was substituted by the bacterial RNA chaperon StpA. The dominant effect of NCp7 is therefore most probably exerted at the level of condensation of the RNA templates, leading to the formation of productive interactions between the nascent DNA and the acceptor template.

Deletion mutagenesis of the Escherichia coli galactose operon promoter region.

Using recombinant DNA technology we have created a series of progressively longer deletions both upstream and downstream from the Escherichia coli galactose operon regulatory region. The effects of these lesions on expression of the two overlapping galactose promoters have been quantitated after DNA fragments carrying these deletions were cloned in a plasmid vector, in which the beta-galactosidase gene could be expressed from the truncated galactose regulatory region. The results allow us to determine which sequences are necessary for the activity of the two promoters. Our results show that for the P1 promoter, which is controlled by the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP), the sequence necessary for full activity starts 56 base-pairs upstream from the transcription initiation point. In contrast, for the P2 promoter, which functions in the absence of cAMP-CRP, the crucial sequence extends to only 39 base-pairs upstream from the transcription start. Deletions that cut into these sequences cause reductions in promoter strength, although some promoter activity is observed even when the "-35 region" of both P2 and P1 are deleted. Analysis of deletions originating downstream from the regulatory region shows that the elimination of the P1 and P2 Pribnow box sequences leads to loss of promoter activity. However, sequences downstream from the P1 start can be replaced without affecting the activity of either promoter. Finally examination of DNA fragments containing total deletions of both galactose promoters allows us to confirm that the flanking sequences contain no significant promoter activity and that the P1 and P2 promoters are principally responsible for galactose operon expression in vivo.

Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon.

The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions -84.5, -74 and -63, with respect ot the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.

DNA curvature controls termination of plus strand DNA synthesis at the centre of HIV-1 genome.

In vivo and in vitro, reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) terminates plus strand synthesis at the centre of the viral genome. The central termination sequence (CTS) contains curved DNA fragments located upstream of each terminator site. Two different models, relying either on the A-tract or general sequence roll assumptions, were used to predict the extent and the direction of this curvature as well as to design mutants, which abolished it. Straightening of each curved element abolished termination at the site located immediately downstream from the curvature. When synthesis was performed on the other strand and in the opposite direction, the two curved elements C1 and C2 associated with the two termination sites Ter1 and Ter2, led again to termination of DNA synthesis. Therefore, termination occurred as a nascent bent duplex was synthesized within the template primer binding cleft of RT, even when putative strand-specific motifs have been removed by the inversion. Computation of DNA paths upstream of other known arrest sites suggested that this feature was of general relevance for termination. At the CTS, termination occurred more precisely at the 3' end of an AnTm motif (n + m = 7). The possible structures, adopted by this motif, are discussed and confronted with the present crystallographic and biochemical data obtained on HIV-1 RT-DNA interactions and on HIV-1 RT processivity.

Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter.

CRP-cAMP was shown to activate transcription initiation at the Escherichia coli lac promoter in vitro as a result of two separate effects. An indirect component of the activation resulted from an enhancement of the fraction of promoters productively bound by RNA polymerase. This effect was due largely to CRP-cAMP repression of RNA polymerase binding to an overlapping site (lac P2) within the promoter region. In addition, a direct enhancement of RNA polymerase binding at the principal lac promoter (lac P1) was found. The combination of indirect and direct activation by CRP-cAMP was suggested to be responsible for the large activation observed in vivo. Promoter strength parameters were also determined for the L8, UV5 and Ps promoters. The effect of CRP-cAMP on these mutant promoters was shown to be consistent with the activation mechanism deduced for the lac wild-type promoter. DNA supercoiling enhanced the promoter strength of the lac wild-type and UV5 promoters. The combination of supercoiling and CRP-cAMP was necessary for optimal promoter strength for the lac wild-type promoter.

The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase.

Time-resolved laser UV irradiation and controlled proteolysis have been used to study the sequential recognition of the lac UV5 promoter by Escherichia coli RNA polymerase. Local rearrangements in the DNA, the appearance of intimate protein-DNA contacts, and structural changes within the sigma subunit together provide specific signatures that define major species populated during this process. At 22 degreesC, a first closed complex is characterised by a transient conformational change in the sigma subunit and by a distortion in the -35 region. Subsequently, direct contacts at -34 and at positions -8, -5 and -3 on the non-template strand appear prior to DNA strand separation. The contact in the -35 consensus region involves only the sigma subunit. This intermediate possesses different structural parameters from that formed by quenching open complexes from 37 degreesC to 14 degreesC. Sigma thus appears as the principal partner acting during promoter recognition, a strongly coupled process involving two major intermediates only.

HIV-1 reverse transcription. A termination step at the center of the genome.

During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication.