DNA trajectory in the Gal repressosome

Semsey, Szabolcs; Tolstorukov, Michail Y.; Virnik, Konstantin; Zhurkin, Victor B.; Adhya, Sankar

doi:10.1101/gad.1209404

Cited by 40 publications

(43 citation statements)

References 40 publications

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“…Up to now, the lac-gal regulon and the gal operon have been reported in Proteobacteria and Firmicutes. The molecular regulation of the gal operon in Proteobacteria such as E. coli is well known (Roy et al, 2004;Semsey et al, 2002Semsey et al, , 2004Semsey et al, , 2006, but the study of Firmicute gal operons is considered to be uncharted territory. Firmicutes are broadly divided into two classes, bacilli and clostridia.…”

Section: Introductionmentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 6C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

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Section: Introductionmentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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“…Prokaryotic and eukaryotic regulatory complexes involving short DNA loops (12)(13)(14)(15)(16)(17)(18) place strong constraints on the helical twist of the looped DNA (2). For two DNA-bound proteins to interact when they are separated along the DNA, the proteinbinding sites need to occur on mutually compatible faces of the DNA double helix.…”

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confidence: 99%

DNA twisting flexibility and the formation of sharply looped protein–DNA complexes

Cloutier

Widom

2005

Proc. Natl. Acad. Sci. U.S.A.

199

244

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Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often Ϸ100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89-to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA.activation ͉ gene regulation ͉ repression D ouble-stranded DNA in vivo is often sharply distorted away from its classic B-form conformations. In many prokaryotic gene-regulatory complexes, short stretches of DNA, Ϸ100 bp in length, are bent sharply into circular or antiparallel (U-shaped or teardrop-shaped) loops (1, 2). Looping allows synergy between proteins bound at distant DNA sites (3) and decreases the statistical noise in their occupancy (4). DNA looping is also important in eukaryotic regulatory systems (5-7). A striking recent example is the discovery of ligand-dependent DNA looping by the RXR receptor, which may play an important regulatory role at as many as 172 different locations, genome wide, in the mouse (8). In addition, most (Ϸ75-80%) of the length of eukaryotic genomic DNA is sharply bent into nucleosomes (80-bp superhelical loops) (9), which regulate the accessibility and proximity of other DNA-functional sites (10, 11).Prokaryotic and eukaryotic regulatory complexes involving short DNA loops (12-18) place strong constraints on the helical twist of the looped DNA (2). For two DNA-bound proteins to interact when they are separated along the DNA, the proteinbinding sites need to occur on mutually compatible faces of the DNA double helix. This requirement is satisfied by a set of lengths for the intervening DNA that differ from one another by integral multiples of the DNA helical repeat, Ϸ10.5 bp. When the exact length of the intervening DNA in such complexes is suboptimal, the DNA may be under-or overtwisted to allow the protein-protein interaction. The DNA helical twist is altered in other biological systems as well, most notably in the nucleosome, in which the wrapped DNA is under-or overtwisted for most of its length (9).DNA bending and twisting deformations that are required for protein-DNA complex formation come at a cost in free energy, which contributes importantly to the net stabilities and functions of the resulting complexes. For these reasons, the inherent bendability and twistability of DNA itself have been a focus of experimental and theoretical investigation. Classic studies revealed double-st...

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“…The dimers could also participate in replication inhibition without contacting DNA by serving as a bridge between bound monomers (41). Handcuffing then may stabilize dimer interactions as has been found in other systems (14)(15)(16)(17). The role of handcuffing then can be to mechanically execute the homeostatic dynamics inherent to the RepA monomer-dimer competition.…”

Section: Discussionmentioning

confidence: 99%

“…To keep titration from counteracting autorepression, it was proposed that the titrated initiators pair with promoter-bound initiators and thereby help maintain the repression (12). This mechanism, now called handcuffing (13), is common among transcriptional repressors that loop DNA, where the titrated repressors, instead of relieving autorepression, actually increase it (14)(15)(16)(17).…”

mentioning

confidence: 99%

Multiple homeostatic mechanisms in the control of P1 plasmid replication

Das

Valjavec-Gratian

Basuray

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Many organisms control initiation of DNA replication by limiting supply or activity of initiator proteins. In plasmids, such as P1, initiators are limited primarily by transcription and dimerization. However, the relevance of initiator limitation to plasmid copy number control has appeared doubtful, because initiator oversupply increases the copy number only marginally. Copy number control instead has been attributed to initiator-mediated plasmid pairing (''handcuffing''), because initiator mutations to handcuffing deficiency elevates the copy number significantly. Here, we present genetic evidence of a role for initiator limitation in plasmid copy number control by showing that autorepression-defective initiator mutants also can elevate the plasmid copy number. We further show, by quantitative modeling, that initiator dimerization is a homeostatic mechanism that dampens active monomer increase when the protein is oversupplied. This finding implies that oversupplied initiator proteins are largely dimeric, partly accounting for their limited ability to increase copy number. A combination of autorepression, dimerization, and handcuffing appears to account fully for control of P1 plasmid copy number.autorepression ͉ DNA replication control ͉ homeostatic control ͉ plasmid copy number control

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DNA trajectory in the Gal repressosome

Cited by 40 publications

References 40 publications

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

DNA twisting flexibility and the formation of sharply looped protein–DNA complexes

Multiple homeostatic mechanisms in the control of P1 plasmid replication

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