Hai Young Wu

Long-Range Interaction between Two Promoters: Activation of the leu.500 Promoter by a Distant Upstream Promoter

The leu-500 mutation can be suppressed in S. typhimurium topA. Previous studies have demonstrated that the plasmid-borne leu-500 minimal promoter cannot be activated in topA mutants unless adjacent (< 250 bp) transcription occurs away from the leu-500 promoter (short-range promoter interaction). To search for a potential upstream promoter responsible for activation of leu-500 in the chromosomal context, we have identified the ilvlH promoter, located 1.9 kb upstream of leu-500 (long-range promoter interaction). Different from short-range promoter interaction, which is abolished by DNA sequence insertions, the long-range promoter interaction is mediated by the intervening DNA sequence. These studies suggest that the long-range interaction between a pair of divergently arrayed promoters is probably mediated by a complex process involving relay of DNA supercoiling by the DNA sequence located between the two promoters.

LeuO Protein Delimits the Transcriptionally Active and Repressive Domains on the Bacterial Chromosome

LeuO protein relieves bacterial gene silencer AT8-mediated transcriptional repression as part of a promoter relay mechanism found in the ilvIH-leuO-leuABCD gene cluster. The gene silencing activity has recently been characterized as a nucleoprotein filament initiated at the gene silencer. In this gene locus, the nucleoprotein filament cis-spreads toward the target leuO promoter and results in the repression of the leuO gene. Although the cis-spreading nature of the transcriptionally repressive nucleoprotein filament has been revealed, the mechanism underlying LeuO-mediated gene silencing relief remains unknown. We have demonstrated here that LeuO functions analogously to the eukaryotic boundary element that delimits the transcriptionally active and repressive domains on the chromosome by blocking the cis-spreading pathway of the transcriptionally repressive heterochromatin. Given that one LeuO-binding site is positioned between the gene silencer and the target promoter, the simultaneous presence of a second LeuO-binding site synergistically enhances the blockade, resulting in a cooperative increase in LeuO-mediated gene silencing relief. A known DNA loop-forming protein, the lac repressor (LacI), was used to confirm that cooperative protein binding via DNA looping is responsible for the blocking synergy. Indeed, a distal LeuO site located downstream cooperates with the LeuO sites located upstream of the leuO gene, resulting in synergistic relief for the repressed leuO gene via looping out the intervening DNA between LeuO sites in the ilvIH-leuO-leuABCD gene cluster.

Transcription-driven DNA supercoiling and gene expression control.

DNA supercoiling plays important roles in gene expression regulation, although, the underlying mechanisms whereby DNA supercoiling modulates gene expression remain elusive. The fact that the transcription process itself generates DNA supercoiling has further complicated the issue. Transcription-driven DNA supercoiling is local and transient. Such a DNA supercoiling effect is likely to play important roles in controlling complex gene expression regulation. Using the suppression of the leu-500 mutation in Salmonella typhimurium topA mutants as a model system, we put forward our view of the effects of transcription-driven DNA supercoiling on gene expression control.

A 72-Base Pair AT-rich DNA Sequence Element Functions as a Bacterial Gene Silencer

We have previously demonstrated that sequential activation of the bacterial ilvIH-leuO-leuABCD gene cluster involves a promoter-relay mechanism. In the current study, we show that the final activation of the leuABCD operon is through a transcriptional derepression mechanism. TheleuABCD operon is transcriptionally repressed by the presence of a 318-base pair AT-rich upstream element. LeuO is required for derepressing the repressed leuABCD operon. Deletion analysis of the repressive effect of the 318-bp element has led to the identification of a 72-bp AT-rich (78% A+T) DNA sequence element, AT4, which is capable of silencing a number of unrelated promoters in addition to the leuABCD promoter. AT4-mediated gene silencing is orientation-independent and occurs within a distance of 300 base pairs. Furthermore, an increased gene-silencing effect was observed with a tandemly repeated AT4 dimer. The possible mechanism of AT4-mediated gene silencing in bacteria is discussed.

Suppression of leu-500 mutation in topA+ Salmonella typhimurium strains. The promoter relay at work.

Suppression of leu-500 mutation inSalmonella typhimurium topA − strains has been one of the most fascinating examples for the DNA supercoiling effect on transcription initiation control. Previous studies have indicated possible involvement of transcription-driven DNA supercoiling in the activation of the leu-500 promoter intopA − strains. Our recent studies have shown that ilvIH transcription activity located 1.9 kilobase pairs upstream is the initial supercoiling signal forleu-500 activation via a promoter relay mechanism. In the present communication, we show that the ilvIH transcription activity-initiated promoter relay can result in leu-500activation in topA + strains. In addition, suppression of the chromosomal leu-500 mutation correlates with the transcription activities of ilvIH andleuO rather than the TopA level in thetopA + strain. It appears that theleu-500 suppression in a topA −strain is due to the constant ilvIH transcription activity.

LeuO-mediated Transcriptional Derepression

To understand the coordination of gene expression in the Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster, we had previously identified a 72-bp AT-rich (78% A+T) DNA sequence element, AT4, which was capable of silencing transcription in a promoter nonspecific manner. LeuO protein provided in trans relieved (derepressed) AT4-mediated gene silencing (transcriptional repression), but underlying mechanisms remained unclear. In the present communication, the 72-bp DNA sequence element is further dissected into two functional elements, AT7 and AT8. LeuO binds to the 25-bp AT7, which lies closest to the leuO promoter in the AT4 DNA. After deletion of the AT7 DNA sequence responsible for LeuO binding from AT4, the remaining 47-bp AT-rich (85% A+T) DNA sequence, termed AT8, retains the full bi-directional gene-silencing activity, which is no longer relieved by LeuO. LeuO-mediated transcriptional derepression is restored when the LeuO binding site, AT7, is placed within close proximity to the gene silencer AT8. As a pair of functionally coupled transcription elements, the presence of an equal copy number of AT7 and AT8 within proximity is important for the transcription control. The characterization provides clues for future elucidation of the molecular details whereby LeuO negates the gene-silencing activity.

Topological approaches to studies of protein-mediated looping of DNA in vivo.

Publisher Summary This chapter presents topological approaches for studying the protein-mediated looping of DNA in vivo. It describes methods for studying protein-mediated looping of DNA in vivo using the lac repressor–operator complex as a model system. The tetrameric lac repressor has been shown to bind simultaneously to two operators both in vitro and in vivo . The simultaneous binding of the lac repressor to two operators on the same DNA molecule causes looping of the intervening DNA. Such lac repressor-mediated looping of DNA interferes with template supercoiling during RNA transcription and can cause changes in the supercoiled state of plasmid DNAs. The looping of DNA due to lac repressor binding requires the presence of at least two lac repressor-binding sites on the same molecule. For plasmid DNAs containing only a single lac repressor binding site, only the multimeric forms of the plasmid DNAs can be efficiently looped by the tetrameric lac repressor complex. The preferential supercoiling of the multimeric forms of the plasmid DNAs can, therefore, be a good indication of protein-mediated looping of DNA in vivo .

Genome Organization: The Effects of Transcription-driven DNA Supercoiling on Gene Expression Regulation

The transcriptional outputs of genes are often dependent on their chromosomal localizations. Such positional effects suggest that the architecture of chromatin structures play roles in gene expression regulation. The molecular mechanisms that underlie the position effects of gene expression regulation remain unclear. In particular, the role of DNA supercoiling that plays in this level of transcriptional control is absent. The fact that the transcription process itself generates DNA supercoiling has further complicated the issue and has led to a hypothesis that DNA supercoiling may be involved in coordinating the expression of multiple genes in a region via modulating the chromosomal architecture or directly altering the structures of the DNA elements. This possibility has been built upon the existence of unconstrained DNA supercoiling on the chromosome.