细菌转录起始调控机制*

原核生物同一种群的每个细胞都是和外界环境直接接触的,它们主要通过开启或关闭某些基因的表达来适应环境条件。所以,环境因子往往是调控的效应因子,必须严格调控转录来确保细胞对环境改变做出有效且充分的反应。原核生物基因的表达受多种因素的调控,而对于大多数细菌来说,调控基因表达的关键步骤是启动子识别和RNA聚合酶启动转录。在细菌的细胞中,可以通过调节RNA聚合酶的活性以及改变RNA聚合酶对启动子的结合来优化基因的转录过程以适应不同环境变化。总结了目前已发现的参与细菌细胞转录调节的各类因子,从这些因子对启动子的作用、RNA聚合酶的作用以及两者的相互作用等方面阐述它们调控基因表达的分子机制。总结多种基因调控的作用,加深对转录起始过程的认识,希望能对未来调控转录起始过程来实现目标基因的高效表达和不利基因的抑制表达提供思路,为以后的工业菌株改造提供依据。     

Loop extrusion driven volume phase transition of entangled chromosomes

Experiments on reconstituted chromosomes have revealed that mitotic chromosomes are assembled even without nucleosomes. When topoisomerase II (topo II) is depleted from such reconstituted chromosomes, these chromosomes are not disentangled and form “sparklers,” where DNA and linker histone are condensed in the core and condensin is localized at the periphery. To understand the mechanism of the assembly of sparklers, we here take into account the loop extrusion by condensin in an extension of the theory of entangled polymer gels. The loop extrusion stiffens an entangled DNA network because DNA segments in the elastically effective chains are translocated to loops, which are elastically ineffective. Our theory predicts that the loop extrusion by condensin drives the volume phase transition that collapses a swollen entangled DNA gel because the stiffening of the network destabilizes the swollen phase. This may be an important piece to understand the mechanism of the assembly of mitotic chromosomes.     

Identification of a human T-cell leukemia virus type I tax peptide in contact with DNA

The human T-cell leukemia virus Tax protein directs binding of a host factor, cAMP response element binding protein, to an extended recognition sequence in the proviral promoter. Prior cross-linking experiments have revealed that Tax makes restricted contact with this DNA at two symmetric positions, 14 nucleotides apart on opposite strands of the DNA. Tax lacks a conventional DNA binding domain, and the sequences in Tax that are in contact with DNA have not been previously identified. Analysis of cross-linked peptides now shows that the contact occurs between Tax residues 89 and 110, corresponding to a protease-sensitive linker joining two protein structural domains. The linker assumes a protease-resistant conformation in the cross-linked complex. Point mutations within the linker prevent cross-linking and interfere with Tax function. These data suggest that entry of Tax into the ternary complex may be coupled to folding of an unstructured protein domain, which then makes base-specific contacts with DNA.     

A new method for constructing linker scanning mutants.

A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from -208 to +15.     

The Accidental Ally: Nucleosome Barriers Can Accelerate Cohesin-Mediated Loop Formation in Chromatin

An important question in the context of the three-dimensional organization of chromosomes is the mechanism of formation of large loops between distant basepairs. Recent experiments suggest that the formation of loops might be mediated by loop extrusion factor proteins such as cohesin. Experiments on cohesin have shown that cohesins walk diffusively on the DNA and that nucleosomes act as obstacles to the diffusion, lowering the permeability and hence reducing the effective diffusion constant. An estimation of the times required to form the loops of typical sizes seen in Hi-C experiments using these low-effective-diffusion constants leads to times that are unphysically large. The puzzle then is the following: how does a cohesin molecule diffusing on the DNA backbone achieve speeds necessary to form the large loops seen in experiments? We propose a simple answer to this puzzle and show that although at low densities, nucleosomes act as barriers to cohesin diffusion, beyond a certain concentration they can reduce loop formation times because of a subtle interplay between the nucleosome size and the mean linker length. This effect is further enhanced on considering stochastic binding kinetics of nucleosomes on the DNA backbone and leads to predictions of lower loop formation times than might be expected from a naive obstacle picture of nucleosomes.