Biomedical subjects

M Guérin

Publications and source records attributed to M Guérin.

At least 19 recordsLinked to original sources

PubMed2001

Atherogenic role of elevated CE transfer from HDL to VLDL(1) and dense LDL in type 2 diabetes : impact of the degree of triglyceridemia.

Plasma cholesteryl ester transfer protein (CETP) facilitates intravascular lipoprotein remodeling by promoting the heteroexchange of neutral lipids. To determine whether the degree of triglyceridemia may influence the CETP-mediated redistribution of HDL CE between atherogenic plasma lipoprotein particles in type 2 diabetes, we evaluated CE mass transfer from HDL to apoB-containing lipoprotein acceptors in the plasma of type 2 diabetes subjects (n=38). In parallel, we investigated the potential relationship between CE transfer and the appearance of an atherogenic dense LDL profile. The diabetic population was divided into 3 subgroups according to fasting plasma triglyceride (TG) levels: group 1 (G1), TG<100 mg/dL; group 2 (G2), 100 200 mg/dL. Type 2 diabetes patients displayed an asymmetrical LDL profile in which the dense LDL subfractions predominated. Plasma levels of dense LDL subfractions were strongly positively correlated with those of plasma triglyceride (TG) (r=0.471; P:=0.0003). The rate of CE mass transfer from HDL to apoB-containing lipoproteins was significantly enhanced in G3 compared with G2 or G1 (46.2+/-8.1, 33.6+/-5.3, and 28.2+/-2.7 microg CE transferred. h(-1). mL(-1) in G3, G2, and G1, respectively; P:<0.0001 G3 versus G1, P:=0.0001 G2 versus G1, and P:=0.02 G2 versus G3). The relative capacities of VLDL and LDL to act as acceptors of CE from HDL were distinct between type 2 diabetes subgroups. LDL particles represented the preferential CE acceptor in G1 and accounted for 74% of total CE transferred from HDL. By contrast, in G2 and G3, TG-rich lipoprotein subfractions accounted for 47% and 72% of total CE transferred from HDL, respectively. Moreover, the relative proportion of CE transferred from HDL to VLDL(1) in type 2 diabetes patients increased progressively with increase in plasma TG levels. The VLDL(1) subfraction accounted for 34%, 43%, and 52% of total CE transferred from HDL to TG-rich lipoproteins in patients from G1, G2, and G3, respectively. Finally, dense LDL acquired an average of 45% of total CE transferred from HDL to LDL in type 2 diabetes patients. In conclusion, CETP contributes significantly to the formation of small dense LDL particles in type 2 diabetes by a preferential CE transfer from HDL to small dense LDL, as well as through an indirect mechanism involving an enhanced CE transfer from HDL to VLDL(1), the specific precursors of small dense LDL particles in plasma.

Adult↗

PubMed2001

[Role of anomalies of low density lipoproteins (LDL) in atherogenicity].

Qualitative and quantitative anomalies of low-density lipoproteins (LDL) play a key role in the pathophysiology of atherosclerosis. Such anomalies are characteristics of the atherogenic dyslipidemias which occur most frequently, i.e. primary hypercholesterolemia of phenotype IIA (including familial hypercholesterolemia), combined hyperlipidemias (Type IIB) and hypertriglyceridemia (Type IV). An elevated concentration of circulating LDL occurs either as a result of hepatic overproduction of VLDL particles, the major precursors of LDL, or as a result of delayed catabolism, as occurs when there is a deficit of cellular LDL receptors (e.g. familial hypercholesterolemia), or as a combination of both. The major qualitative anomaly of LDL which results in elevated atherogenicity involves a predominance of small dense LDL, as seen in patients with premature coronary heart disease and equally in combined hyperlipidemia and in hypertriglyceridemia. The mechanism of the formation of these particles is complex and involves the concerted intravascular action of cholesteryl ester transfer protein (CETP), lipoprotein lipase (LPL) and hepatic lipase (HL) on triglyceride-rich precursors of dense LDL Lipid-lowering agents, such as fibrates and statins, act to reduce the atherogenicity of dense LDL by distinct mechanisms, which lead to normalisation of circulating LDL levels and/or to targeted reduction in dense particles of elevated atherogenicity. Indeed, such pharmacological probes have facilitated new insight into the molecular and cellular mechanisms which underlie each of the major forms of atherogenic dyslipidemia.

Arteriosclerosis↗

PubMed2000

The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation.

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.

Basic Helix-Loop-Helix Proteins↗

PubMed1999

In vivo evidence for back and forth oscillations of the transcription elongation complex.

We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock. We show that the complex readblocked within an (ATC/TAG)(n) sequence is in a dynamic equilibrium between upstream- and downstream- translocated conformers. By increasing the strength of the putative RNA-DNA hybrid, the ternary complex is readily trapped in the downstream-translocated conformation, where the melted DNA region is limited to 8 bp. The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5' end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis-acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.

Base Sequence↗

PubMed1999

Neurodegenerative disorders: the role of peroxynitrite.

Inflammatory reaction is thought to be an important contributor to neuronal damage in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and the parkinsonism dementia complex of Guam. Among the toxic agents released in brain tissues by activated cells, we focus attention in this review on peroxynitrite, the product of the reaction between nitric oxide (NO) and superoxide. Peroxynitrite is a strong oxidizing and nitrating agent which can react with all classes of biomolecules. In the CNS it can be generated by microglial cells activated by pro-inflammatory cytokines or beta-amyloid peptide (beta-A) and by neurons in three different situations: hyperactivity of glutamate neurotransmission, mitochondrial dysfunction and depletion of L-arginine or tetrahydrobiopterin. The first two situations correspond to cellular responses to an initial neuronal injury and the peroxynitrite formed only exacerbates the inflammatory process, whereas in the third situation the peroxynitrite generated directly contributes to the initiation of the neurodegenerative process.

Alzheimer Disease↗

PubMed1998

A simple polypyrimidine repeat acts as an artificial Rho-dependent terminator in vivo and in vitro.

In this paper, we present evidence that an efficient Rho-dependent terminator can be created by introducing a simple (AG/TC) n DNA repeat into a transcription unit. The Rho termination activity in vivo and in vitro is dependent on the length and the orientation of the insert. The transcription of at least 30 bp of the (AG/TC) n repeat in the orientation encoding the (rUrC) n sequence on the transcript leads to Rho-dependent termination at a downstream non-terminator site. Our results indicate that the high efficiency of this artificial Rho-dependent terminator is due to optimal interactions between the (rUrC) n RNA sequence and Rho protein. Thus, our findings strongly suggest that an adequate loading site is the primary determinant for Rho termination activity and provide a more defined system for future investigations.

Base Sequence↗

PubMed1998

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

DNA, Superhelical↗

PubMed1998

Characterization of the yeast mitochondria unselective channel: a counterpart to the mammalian permeability transition pore?

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca2+ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a "yeast permeability transition pore."

Adenosine Triphosphate↗

PubMed1998

Investigation of the yeast mitochondrial unselective channel in intact and permeabilized spheroplasts.

The existence of an activity corresponding to the nucleotide-induced Yeast Mitochondria Unselective Channel (YMUC2) of isolated mitochondria was investigated in permeabilized and intact spheroplasts of the baker's yeast Yeast Foam. In nystatin-permeabilized spheroplasts, ATP and GDP-beta-S induced a decavanadate-sensitive stimulation of the respiration only under conditions equivalent to those previously reported for isolated mitochondria (low phosphate concentration, presence of a salt). On intact spheroplasts parallel measurements of respiration rate, [ATP]/[ADP] ratio and mitochondrial transmembrane potential allowed to show that the addition of the glucose analog 2-deoxyglucose decreased the permeability of the inner mitochondrial membrane owing to cellular ATP depletion. This strongly supports the hypothesis that Yeast Mitochondria Unspecific Channel is active in situ and inhibited by cellular [ATP] depletion.

Adenosine Diphosphate↗

PubMed1998

Preferential cholesteryl ester acceptors among triglyceride-rich lipoproteins during alimentary lipemia in normolipidemic subjects.

Triglyceride-rich lipoproteins (TRLs), namely chylomicrons (CMs), VLDL, and their remnants, are implicated in the atherogenic features of postprandial lipemia. In human plasma, cholesteryl ester transfer protein (CETP) mediates the heteroexchange of neutral lipids, ie, triglycerides (TG) and cholesteryl esters (CE), between distinct subpopulations of apoB- and of apoAI-containing lipoproteins. In fasting normolipidemic plasma, CETP plays an antiatherogenic role by promoting preferential CE redistribution from HDL to LDL particles of intermediate subclass with optimal binding affinity for the cellular LDL receptor. While the relative proportions and chemical compositions of donor and acceptor lipoproteins are known to influence CETP activity, elevated levels of TRL present during alimentary lipemia have been proposed to be associated with enhanced CETP activity. To identify the preferential CE acceptor particles among postprandial TRL subfractions, we investigated the effects of a typical Western meal (1200 kcal, 14% protein; 38% carbohydrate; and 48% fat, monounsaturated/polyunsaturated ratio 4:1) on the rates of postprandial CE transfer from HDL to apoB-containing lipoproteins in normolipidemic subjects (n=13). Two hours postprandially, plasma levels of TRL were significantly elevated (140 versus 51 mg/dL at baseline, P=.0001). Total rates of CE transferred (88 +/- 7 microg x h[-1] x mL[-1]) from HDL to apoB-containing lipoproteins were not significantly modified by alimentary lipemia over a period of 8 hours. Quantitatively, LDL accepted 64+/-5 microg CE per hour per milliliter plasma from HDL, whereas CM (Sf>400), VLDL1 (Sf 60 to 400), VLDL2 (Sf 20 to 60), and IDL (Sf 12 to 20) accepted 5+/-3, 16+/-3, 1.4+/-0.3, and 1.5+/-0.2, respectively. Quantitatively, VLDL1 was the major CE acceptor among TRLs (P=.0001); thus, VLDL1, but not CMs, represented the major CE acceptor among TRLs. Qualitatively however, VLDL2 and IDL displayed a higher capacity to accept CE from HDL (51.6+/-4.1 and 46.3+/-2.8 microg CE transferred per hour per milligram lipoprotein, respectively; P<.005) compared with CM, VLDL1, and LDL (12.6+/-2.8, 34.7+/-4.2, and 22.7+/-2.0 microg CE transferred per hour per milligram lipoprotein, respectively). In conclusion, elevated postprandial TRL levels are not associated with enhanced total CE transfer to these particles. Furthermore, the qualitative features of postprandial CE transfer from HDL to CM and VLDL1 were not related to the relative TG content of these particles. The CETP-facilitated enrichment of VLDL1 in CE therefore identifies them as potentially atherogenic particles during the postprandial phase.

Adult↗

PubMed1998

Atherogenic, dense low-density lipoproteins. Pathophysiology and new therapeutic approaches.

It is well established that elevated circulating concentrations of cholesterol-rich, low-density lipoproteins (LDL) represent a major risk factor for the premature development of coronary artery disease. Only recently, however, has attention been drawn to the relationship between the qualitative features of plasma LDL particles and cardiovascular risk, particularly in view of the frequent occurrence of increased levels of dense, small LDL in coronary artery disease patients. Combined hyperlipidaemia, a frequent form of dyslipidaemia which is associated with premature atherosclerosis, is characterized by elevated plasma concentrations of both triglyceride-rich, very-low-density lipoproteins (VLDL) and LDL. In combined hyperlipidaemia patients, small, dense LDL (d 1.04-1.06 g.ml-1) predominate over the light (d 1.02-1.03 g.ml-1) and intermediate (d 1.03-1.04 g.ml-1) LDL subpopulations. Dense LDL are highly atherogenic as a result of their low binding affinity for the LDL receptor, their prolonged plasma half-life and low resistance to oxidative stress. Biological modification of dense LDL is potentiated as a result of retention in the arterial intima upon binding to extracellular matrix components and exposure to oxidative stress, leading to uptake by macrophages with subsequent foam cell formation. Such cholesterol-loaded, macrophage foam cells are active secretory cells, and exert multiple proinflammatory, proatherogenic and prothrombogenic effects during the initiation and progression of atherosclerotic plaques. Indeed, the secretory products of foam cells play a key role in the fragilization of lipid-rich plaques, leading ultimately to plaque rupture and the associated thrombotic complications. As the pharmacological modulation of dense LDL levels is of special interest, representing a new therapeutic approach in the treatment of atherogenic dyslipidaemia, we probed the biological mechanisms which underlie formation of dense LDL particles in combined hyperlipidaemia patients with a fibrate derivate, fenofibrate. Drug treatment (micronized fenofibrate, 200 mg.day-1 for 8 weeks) induced significant reductions in the plasma concentrations of VLDL (-37%; P < 0.005), and of dense LDL (-21.5%; P < 0.05), with simultaneous increase in HDL-cholesterol (+19%; P < 0.0001). An endogenous assay of cholesteryl ester transfer from cardioprotective HDL to atherogenic, apolipoprotein B-containing lipoproteins (VLDL and LDL) revealed marked reduction (-38%) in cholesterol ester transfer from HDL to VLDL upon fenofibrate treatment, whereas no modification in the low rate of cholesteryl ester transfer between HDL and LDL was detected. Simultaneously, however, the LDL profile in combined hyperlipidaemia patients, which is characterized by a predominance of small, dense LDL, was shifted towards the LDL subpopulation of intermediate density and larger size. Particles of the intermediate LDL subclass are avidly bound and degraded by the cellular LDL receptor which represents the major, non-atherogenic pathway for catabolism of LDL-cholesterol. Our findings indicate that the overall mechanism of the fenofibrate-induced modulation of the atherogenic dense LDL profile in combined hyperlipidaemia involves reduction in cholesteryl ester transfer from HDL to VLDL, together with normalization of the intravascular transformation of hepatic VLDL to receptor-active LDL of intermediate density.

Arteriosclerosis↗

PubMed1997

Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.

The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected. Coexpression of Bcl-xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

Apoptosis↗

PubMed1997

Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modulation of gene expression.

In the reaction between trans-diamminedichloroplatinum(II) and single-stranded oligo(2'-O-methyl ribonucleotide)s containing the sequence GNG (N being a nucleotide residue), the 1,3-trans-{Pt-(NH3)2[GNG]} cross-links are formed. The 1,3-intrastrand cross-links are inert within the single-stranded oligonucleotides. By contrast, they rearrange into interstrand cross-links when the platinated oligonucleotides are paired with their complementary RNA strands. The rate of the interstrand cross-linking reaction depends upon the sequence facing the intrastrand cross-links. When the complementary sequences are 5'-CN'C (N' being a nucleotide), the rates are rather slow (T1/2 >/= 3 h at 37 degrees C). The rearrangement of the intrastrand cross-links into interstrand cross-links can be achieved in a few minutes when the triplets facing the intrastrand cross-links are replaced by doublet 5'-UA or 5'-CA. In vitro, the specificity of the cross-linking reaction between a platinated oligo(2'-O-methyl ribonucleotide) and its target sequence (containing the 5'-CA doublet) located within the coding region of Ha-ras mRNA is demonstrated by steric blocking of reverse transcriptase and translation machinery. Within the HBL100ras1 cells, this platinated oligonucleotide binds specifically and irreversibly to the cognate Ha-ras mRNA. It also inhibits the proliferation of the HBL100ras1 cells in a dose-dependent manner. The fast and specific interstrand cross-linking reaction triggered by the formation of a double helix between platinated oligo(2'-O-methyl ribonucleotide)s and RNA enhances the potential of the oligonucleotides which do not induce mRNA cleavage by RNase H, to modulate gene expression by steric blocking of the translation machinery.

Cell Division↗

PubMed1997

Conditions allowing different states of ATP- and GDP-induced permeability in mitochondria from different strains of Saccharomyces cerevisiae.

The effect of ATP and other nucleotides on the respiration of Saccharomyces cerevisiae mitochondria was investigated. It was observed that ATP induced a stimulation of the respiration rate only in the presence of a salt in mitochondria from the baker's yeast Yeast Foam, whereas an ATP-induced stimulation occurred even in the absence of salt in mitochondria from three different laboratory strains. In both cases, the stimulation was related to a collapse of the transmembrane potential, suggesting the opening of ion- and/or proton-conducting pathways. Not only ATP, but also GTP and CTP, induced these pathways. Moreover, a similar stimulation was obtained with GDP and its analog GDP-beta-S. The fact that, as opposed to NTPs, GDP did not induce any non-specific anion channel, allowed us to use it to demonstrate unambiguously that a proton-conducting pathway was opened through the inner mitochondrial membrane of laboratory strains but not of Yeast Foam. Three additional aspects of this nucleotide-induced permeability were investigated. (i) The proton-conducting pathway was insensitive to Mg2+, whereas the anion-conducting pathway was fully inhibited by 4 mM Mg2-. (ii) The proton-conducting pathway of mitochondria isolated from laboratory strains was opened by the action of nucleotides outside the mitochondrion, since it was fully insensitive to (carboxy)atractyloside, and fully active in mitochondria isolated from op1 and delta anc strains. On the other hand, the cation-conducting pathway of Yeast Foam mitochondria was partly sensitive to (carboxy)atractyloside and insensitive to bongkrekic acid, suggesting a role of the conformational state of ANC in this activity. (iii) Both the proton and cation-conducting pathways were inhibited by very low concentrations of vanadate, under conditions where this oxyanion was polymerized to decavanadate: a competitor to nucleotide-binding sites on some enzymes.

Adenosine Triphosphatases↗

PubMed1997

The ATP-induced K(+)-transport pathway of yeast mitochondria may function as an uncoupling pathway.

The effect of the presence of K+ during oxidative phosphorylation measured on isolated yeast mitochondria was dependent on phosphate concentration. At 0.5 mM phosphate, K+ did promote an uncoupling of oxidative phosphorylation, which was prevented by decavanadate, a potent inhibitor of the ATP-induced K(+)-transport pathway. AT 5 mM phosphate, no uncoupling effect of K+ could be evidenced. These data suggest that the ATP-induced K(+)-transport pathway may, under certain conditions, function as an uncoupling pathway of oxidative phosphorylation.

Adenosine Triphosphate↗

PubMed1997

Modulation of the electrophoretic ATP-induced K(+)-transport in yeast mitochondria by delta pH.

The modulation of the ATP-induced K(+)-transport pathway of the yeast inner mitochondrial membrane by delta pH was investigated in two ways. First, the inhibitory effect of phosphate was compared to the effect of other permeant acids, demonstrating that a part of the effect of phosphate was linked to its electroneutral transport down delta pH. However, an additional effect specific for phosphate also occurred inside the matrix. Second, the stimulation of the respiration by ATP in the presence of K+ was compared to the effects of the protonophore ClCCP2 and of the K(+)-ionophore valinomycin. Quite unexpectedly, the effect of ATP looked more like the effect of ClCCP than the effect of valinomycin. This and previous results (Manon et al., Biochimica et Biophysica Acta 1231, 282-288 (1995)) show that the rate of electrophoretic K(+)-entry is limited by the rate of electroneutral K(+)-exit via the K+/H+ exchange and is therefore delta pH-dependent.

Adenosine Triphosphate↗

PubMed1996

Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphorylation complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) or colorless native polyacrylamide gel electrophoresis (CN-PAGE) allowed separation of the oxidative phosphorylation complexes of yeast mitochondria. These complexes were characterized by specific staining related to their enzymatic activity. Solubilization of mitochondria by different nonionic detergents such as Triton X-100, dodecyl maltoside, Nonidet P-40, Lubrol, octyl glucoside, or Hecameg led to the separation of F1-FO ATPase complexes exhibiting distinct apparent molecular masses related to different contaminating proteins and lipids. All these different forms were active in ATP hydrolysis as revealed directly on the gel. Analysis of the subunit composition of these complexes was carried out by a two-dimensional Tricine-SDS-PAGE and showed that the purest F1-FO ATPase complex was obtained with Lubrol, whereas with Hecameg and octyl glucoside, only the F1 part of ATPase was solubilized.

Chemistry Techniques, Analytical↗

PubMed1996

High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand.

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center. Furthermore, our analyses reveal protein contacts with the non-transcribed strand within the arrested ternary complex. Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non-transcribed strand is an important regulatory element.

Acetaldehyde↗