Kinetic studies and structural models of the association of E. coli sigma(70) RNA polymerase with the lambdaP(R) promoter: large scale conformational changes in forming the kinetically significant intermediates

The kinetics of interaction of Esigma(70) RNA polymerase (R) with the lambdaP(R) promoter (P) were investigated by filter binding over a broad range of temperatures (7.3-42 degrees C) and concentrations of RNA polymerase (1-123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I(2), RP(o)) are single-exponential with first order rate constant beta(CR). Interpretation of the polymerase concentration dependence of beta(CR) in terms of the three step mechanism of open complex formation yields the equilibrium constant K(1) for formation of the first kinetically significant intermediate (I(1)) and the forward rate constant (k(2)) for the conformational change converting I(1) to the second kinetically significant intermediate I(2): R + P-->(K(1))<--I(1)(k(2))-->I(2). Use of rapid quench mixing allows K(1) and k(2) to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60 bp) interface formed in I(1), its relatively small binding constant K(1) at 37 degrees C at this [salt] (approximately 6 x 10(6) M(-1)) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k(a)[DeltaC(o,++)(a)= -1.5(+/-0.2)kcal K(-1)], now shown to originate directly from formation of I(1) [DeltaC(o)(1)= -1.4(+/-0.3)kcal K(-1)] rather than from the formation of I(2) as previously proposed. The isomerization I(1)-->I(2) exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy [E(act)(2)= 34(+/-2)kcal]. This kinetic signature suggests that formation of the transition state (I(1)-I(2)++ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I(1) involves coupled folding of unstructured regions of polymerase (beta, beta' and sigma(70)) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of sigma(70) and possibly domain 1 of beta induce a kink at the -11/-12 base pairs of the lambdaP(R) promoter which places the downstream DNA (-5 to +20) in the jaws of the beta and beta' subunits of polymerase in I(1). These early interactions of beta and beta' with the DNA downstream of position -5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.     

Site-specific DNA binding of nuclear factor I: effect of the spacer region.

Nuclear factor I (NFI) is a site-specific DNA binding protein required for the replication of adenovirus type 2 DNA in vitro and in vivo. To study sequence requirements for the interaction of NFI with DNA, we have measured the binding of the protein to a variety of synthetic sites. Binding sites for NFI (FIB sites) were previously shown to contain a consensus sequence composed of 2 motifs, TGG (Motif 1), and GCCAA (Motif 2), separated by a 6 or 7bp spacer region. To assess conserved sequences in the spacer region and flanking sequences which affect NFI binding, we have isolated clones from oligonucleotide libraries that contain the two motifs flanked by 3 degenerate nucleotides and separated by degenerate spacer regions of 6 or 7 nucleotides. With a 6bp spacer region, a strong bias exists for a C or A residue in the first position of the spacer. Sites with a 7bp spacer region contain a G and C or A residue at the first and second positions, respectively, of the spacer, but also possess conserved residues at other positions of the site.     

Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein

Homologous pairing of single strands with duplex DNA promoted by recA protein occurred without a lag only when the protein was preincubated with ATP and single-stranded DNA. The rate-limiting presynaptic interaction of recA protein and single strands showed a high temperature coefficient: it proceeded 30 times more slowly at 30 degrees C than at 37 degrees C, whereas synapsis showed a normal temperature coefficient. Thus, the presynaptic phase could be separated experimentally from the rest of the reaction by preincubation of single strands with recA protein and ATP at 37 degrees C, followed by a shift to 30 degrees C before double-stranded DNA was added. The presynaptic phase was an order of magnitude more sensitive to inhibition by ADP than was subsequent strand exchange. Presynaptic complexes that were formed at 37 degrees C decayed only slowly at 30 degrees C, but Escherichia coli single strand binding protein caused complexes to form rapidly at 30 degrees C which indicates that single strand binding protein accelerated the rate of formation of complexes. Preincubation synchronized the initial pairing reaction, and further revealed the rapid formation of nascent heteroduplex DNA 250-300 base pairs in length.     

A general upstream binding factor for genes of the yeast translational apparatus.

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in D. melanogaster DNA were studied by electron microscopy (EM). The results of these studies differ from the previously published observations regarding the number, distribution and the size of inverted repeats (ir) present in DNA. -1. In contrast to the previous published observation almost all (96%) of the ir were found in crowded clusters. The DNA strands with clustered palindromes contained 2-21 palindromes (4-42 ir), with an average of 7.25 palindromes (14.5 ir) per strand. No correlation could be found between the length of the DNA strands and the number of ir per strand. -2, Also contrary to some previously published results, most (80%) of the ir formed on renaturation unlooped palindromes and these were always clustered. Looped palindromes (hairpins, formed by renaturation of ir separated by a non-homologous sequence long enough to be seen in EM as single-stranded loop) were found 1-2 per DNA strand, as part of clusters or as solitary palindromes in a DNA strand. The average spacing length (inside clusters) between centers of all palindromes was 2.349 kb, and between centers of looped palindromes 7.6 kb. - 3. The length of the ir was found to be smaller than documented in most of the previously published results. The majority, 80-90%, of the ir found in the unlooped and looped palindromes, respectively, belonged to one main-size class with a range of 30-210 bp and an average length of 100 bp, but longer ir were also observed. The average length of the ir in unlooped palindromes was 124 bp, in looped 244 bp, and the total average was 148 bp - 4. It was calculated that there are about 30,000 palindromes (60,000 ir) in the D, melanogaster genome, of which about 24,000 are unlooped and 6,000 looped, with the spacing between centers of all palindromes averaging about 4.4 kb in length.     

Study of the interaction of DNA with cisplatin and other Pd(II) and Pt(II) complexes by atomic force microscopy.

Modifications in the structure of a 260 bp DNA (hlyM) fragment from Escherichia coli caused by interaction with Pd(II) and Pt(II) complexes were studied. Cisplatin and transplatin [cis- and trans-PtCl2(NH3)2 respectively], Pt2Cl2(Spym)4 (Spym = 2-mercaptopyrimidine anion), Pd-famotidine and Pt-famotidine were incubated with DNA for 24 h at 37 degrees C and then observed with an atomic force microscope. Atomic force microscopy (AFM) provides the opportunity for nanometer resolution in research on the interaction between nucleic acids and metal complexes. The complexes induced noticeable changes in DNA topography according to their different characteristics and structure. In the case of cisplatin a shortening in DNA strands was observed. Transplatin and Pt2Cl2(Spym)4 caused shortening and compaction, whilst an aggregation of two strands was observed for the Pt-famotidine compound but not for the Pd-famotidine compound or the metal-free famotidine.     

Helical periodicity of (PNA)2.(DNA) triplexes in strand displacement complexes.

To study the helical structure in a P-loop formed by an invasion of oligopyrimidine peptide nucleic acid (PNA) into DNA duplex, bent DNA fragments containing a homopurine.homopyrimidine sequence between two bent DNA loci were prepared. As the spacer DNA length between the two bent loci varied by 1 bp over one helical turn, the electrophoretic mobility, reflecting the overall extent of DNA bending, was modulated sinusoidally in non-denaturing 5% polyacrylamide gel. When the bent DNA fragments differing in the spacer DNA length were preincubated with an oligopyrimidine PNA, the gel mobilities were changed due to a P-loop formation. By analyzing the gel mobility data with variations of the P-loop size, average helical parameters at the P-loop structure were determined. (PNA)2. (DNA) triplex within a P-loop had the helical periodicities of 15. 6(0.2) bp per turn at 20 degrees C and 17.4(0.7) bp per turn at 10 degrees C. In addition, the results indicate that a helical unwinding by 57(7) degrees at 20 degrees C and 37(13) degrees at 10 degrees C is present at the two junctions between a P-loop and its adjacent DNA duplex.     

Expression of the mitochondrial genome in HeLa cells : XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA

HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in Mus musculus DNA were examined by electron microscopy (EM). The majority (85%) of palindromes were found to be clustered in about one half of the DNA strands. The rest of the DNA strands were seen with a solitary looped structure. — The unlooped palindromes constituted 53% of all palindromes and were always clustered. There was a significant reduction in the number of unlooped palindromes in comparison to D. melanogaster DNA (Biezunski, 1981) and as a result the palindrome clusters were smaller and contained 2–8 palindromes [4–16 inverted repeats (ir)] per DNA strand. The looped palindromes had a wide and regular distribution with spacing lengths similar to those found in D. melanogaster DNA, and showed some periodicity. The average spacing between centers of all palindromes (inside a cluster) was 4.325 kb, and between centers of looped palindromes 8.544 kb. — The lengths of the ir of unlooped and looped palindromes were grouped (similar to D. melanogaster DNA) in one size-class with a range of 30–240 bp and an average length of 130 bp. Longer ir were also observed and the average length of ir in unlooped palindromes was 186 bp, in looped 588 bp, and the total average length was 375 bp. — It was calculated that there are about 224,000–320,000 palindromes (ir pairs) in the mouse genome, with the spacing between centers of all palindromes about 13-9 kb in length. — In high molecular weight mouse DNA, complex looped structures composed of rows of 5–8 looped palindromes one on top the other, formed by renaturation of multiple ir, were observed. It is suggested, that clustered repetitive sequences, in direct and inverted orientation, might be of one family and homologous to one another, and be able to reassociate, in vitro and in vivo, into structures of different forms, which could function as binding sites for various regulatory proteins during mouse development.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.     

A cloning vehicle suitable for strand separation

A new plasmid has been constructed which contains a poly(A) : poly(dT) duplex segment of length approx. 100 base pairs (bp) inserted into the PvuII site of pBR322. This plasmid, pKH47, has all the other restriction sites of pBR322 available for insertion of foreign DNA, and has the same drug resistance genes as does the parental plasmid. The complementary strands of the linearized denatured plasmid DNA can be separated rapidly an efficiently by affinity chromatography with oligo(dA)- and oligo(dT)-cellulose columns in series. More than 90% of the input DNA is recovered as separated strands which can be annealed to form full length double-stranded molecules. One of the applications of the plasmid is to prepare separated complementary strands for sequencing by the chain-terminator technique using DNA primers. This application is illustrated by a sequencing example for a Drosophila DNA insert carrying a tRNA gene.     

The role of the loxP spacer region in P1 site-specific recombination.

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.