Direct Characterization of Transcription Elongation by RNA Polymerase I.

RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo.

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells.

Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops--that aid or inhibit enhancer-promoter contact--are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. Nested loops assist each other's formation consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other's formation, thus providing clear support for the loop domain model for insulation. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

The lytic-lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA.

The effect of nonspecific binding of lambda repressor on DNA looping dynamics.

The λ repressor (CI) protein-induced DNA loop maintains stable lysogeny, yet allows efficient switching to lysis. Herein, the kinetics of loop formation and breakdown has been characterized at various concentrations of protein using tethered particle microscopy and a novel, to our knowledge, method of analysis. Our results show that a broad distribution of rate constants and complex kinetics underlie loop formation and breakdown. In addition, comparison of the kinetics of looping in wild-type DNA and DNA with mutated o3 operators showed that these sites may trigger nucleation of nonspecific binding at the closure of the loop. The average activation energy calculated from the rate constant distribution is consistent with a model in which nonspecific binding of CI between the operators shortens their effective separation, thereby lowering the energy barrier for loop formation and broadening the rate constant distribution for looping. Similarly, nonspecific binding affects the kinetics of loop breakdown by increasing the number of loop-securing protein interactions, and broadens the rate constant distribution for this reaction. Therefore, simultaneous increase of the rate constant for loop formation and reduction of that for loop breakdown stabilizes lysogeny. Given these simultaneous changes, the frequency of transitions between the looped and the unlooped state remains nearly constant. Although the loop becomes more stable thermodynamically with increasing CI concentration, it still opens periodically, conferring sensitivity to environmental changes, which may require switching to lytic conditions.

Dividing a supercoiled DNA molecule into two independent topological domains.

Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.

Single-molecule approaches to probe the structure, kinetics, and thermodynamics of nucleoprotein complexes that regulate transcription.

Single-molecule experimentation has contributed significantly to our understanding of the mechanics of nucleoprotein complexes that regulate epigenetic switches. In this minireview, we will discuss the application of the tethered-particle motion technique, magnetic tweezers, and atomic force microscopy to (i) directly visualize and thermodynamically characterize DNA loops induced by the lac, gal, and lambda repressors and (ii) understand the mechanistic role of DNA-supercoiling and DNA-bending cofactors in both prokaryotic and eukaryotic systems.

Integration host factor alters LacI-induced DNA looping.

The integration host factor protein of Escherichia coli, which sharply bends DNA at specific sites and non-specifically compacts the bacterial genome, can also alter looping of DNA in an artificial system based on the lactose repressor protein of E. coli. In single molecule experiments, we show that both specific bending and non-specific compaction alter LacI-mediated looping of DNA. Our results highlight the subtle regulatory roles that proteins, which confer structure upon DNA, might have in controlling DNA transcription and other processes in which the conformation of DNA determines the binding and activity of processive enzymes.

DNA compaction by the nuclear factor-Y.

The nuclear factor-Y (NF-Y), a trimeric, CCAAT-binding transcriptional activator with histone-like subunits, was until recently considered a prototypical promoter transcription factor. However, recent in vivo chromatin immunoprecipitation assays associated with microarray methodologies (chromatin immunoprecipitation on chip experiments) have indicated that a large portion of target sites (40%-50%) are located outside of core promoters. We applied the tethered particle motion technique to the major histocompatibility complex class II enhancer-promoter region to characterize i), the progressive compaction of DNA due to increasing concentrations of NF-Y, ii), the role of specific subunits and domains of NF-Y in the process, and iii), the interplay between NF-Y and the regulatory factor-X, which cooperatively binds to the X-box adjacent to the CCAAT box. Our study shows that NF-Y has histone-like activity, since it binds DNA nonspecifically with high affinity to compact it. This activity, which depends on the presence of all trimer subunits and of their glutamine-rich domains, seems to be attenuated by the transcriptional cofactor regulatory factor-X. Most importantly NF-Y-induced DNA compaction may facilitate promoter-enhancer interactions, which are known to be critical for expression regulation.

Diffusion of light-harvesting complex II in the thylakoid membranes.

The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions.