Observation by fluorescence microscopy of transcription on single combed DNA

Abstract

Molecular combing is a powerful procedure for aligning a large array of DNA molecules onto a surface. This technique usually leads to an overstretching of about 150% of the molecules' contour length. By changing the magnitude of capillary forces during the combing process, we were able to reduce the relative extension of the DNA molecules. Thus we achieved combing of T7 DNA with an extension close to its molecule contour length. We checked the ability of combed DNA to interact with DNA binding proteins. Using the T7 bacteriophage transcription system, we investigated the transcription activity of RNA polymerase on combed DNA by direct visualization of newly synthesized fluorescent RNAs. Our experiments show that no transcription activity occurs on overstretched DNA molecules, whereas we observe a transcription activity for nonoverstretched molecules. This activity is observed both in multiple initiation experiments and for one immobilized T7 RNA polymerase per promoter. These results open possibilities for the study of single enzyme actions on combed DNA by optical methods. T ranscription is a fundamental process in gene expression that allows the regulation of cellular adaptation and differentiation. This function is carried out by RNA polymerases (RNAPs) that produce an RNA copy of a given DNA strand (1). The interaction between the DNA and the RNAP is complex because RNAP must recognize a promoter, a sequence-specific region of doublestranded DNA before polymerization. After isomerization of the nucleoprotein complex, resulting in local melting of the double helix, the enzyme transcribes DNA into RNA following the doublestranded DNA until it reaches a terminator (2). Motion is therefore part of the intrinsic activity of RNAP (3). Having the possibility to follow and visualize the movement of an RNAP on the DNA template will open a new area of investigation for understanding the mechanisms of transcription and its regulation.Our goal is to detect the activity of RNAP along a DNA molecule during the transcription process. Several groups have reported single molecule investigation of the transcription process (4-9). Because we use fluorescence microscopy techniques, we cannot use DNA in its normal aqueous solution state, which is a Brownian fluctuating coil. Therefore the first steps we have to perform are to stretch the DNA molecule to avoid conformational fluctuations and to hinder its Brownian motion. Several techniques permit researchers to obtain both immobilized and stretched DNA molecules. The techniques developed so far are micromanipulation with optical or magnetic tweezers (10-14), elongation in a flow (15) or in an electric field (16), and molecular combing (17). Although micromanipulation has provided much insight it has an inherent disadvantage because the observation is limited to one molecule at a time and large statistics are difficult to obtain. By contrast, molecular combing is of particular interest because it allows direct observation of a large array of immobilized and aligned DNA by fluores...

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…By tracking a tagged RNAP itself, or by monitoring the incorporation of fluorescent nucleotides into an RNA chain, the processes of promoter search or elongation can be studied with minimal perturbation (19)(20)(21)(22)(23). Structural rearrangements of the TEC that occur during the transcription cycle can also be monitored using fluorescence (Förster) resonance energy transfer (FRET) (Figure 2) (6,24,25).…”

Sequence information can be obtained from single DNA molecules

“…Other proposals turn to nature for inspiration and seek to combine optical techniques with enzymes that have been fine-tuned by evolution to operate as machines that assemble and disassemble DNA with inherent single-base resolution (6,11,12). Although there have been single molecule studies of DNA polymerase (16, 17), RNA polymerase (18,19), and exonuclease (20, 21), measuring the activity of these enzymes with single-base resolution has been an elusive goal. We took advantage of the exquisite discrimination and fidelity of DNA polymerase to image sequence information in a single DNA template as its complementary strand is synthesized.…”

Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

“…It was noted, however, that transcription efficiency dropped when too much charged silane was conjugated to the surface since higher charge densities promoted DNA overstretching, which has been previously shown to inhibit transcription activity [15]. We determined that stretching template molecules to 70% to 80% of their full contour length was optimal for identifying ECs with confidence while keeping sufficient transcription efficiency, which was the case on 150 trimethylsilane surfaces that we determined to use in subsequent experiments.…”

“…An important consideration in the development and use of single molecule arrays is preservation of the biochemical competence of deposited analyte molecules. In this regard, a method that used harsh buffer pH conditions produced overstretched DNA molecules to 150% of their contour length, which inhibited transcription activities [15]. In contrast, the Optical Mapping (OM) system creates massive arrays of large DNA molecules that are stretched by fluid flows in microchannels.…”