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Crystal structure of core polymerase

Posted by star on 2018-10-19 00:50:46

    Although the core polymerase crystal structure provides considerable information about RNA polymerase structure, it provides no direct information about interactions that exist among core polymerase, σ factor, and DNA. Darst and coworkers addressed these and related structural issues by preparing crystals that contained the Trq holoenzyme bound ta a synthetic DNA with a fork-junction sequence (the junction between double-stranded DNA and single-stranded DNA in the transcription bubble).

    The DNA molecule they synthesized for this purpose has a double-stranded -35 box, a mostly open -10 box, and an extended -10 element. The crystal structure of the RNA polymerase holoenzyme· fork-junction DNA complex, which appears to resemble the open complex. The σ70 factor assumes a different conformation when bound to core polymerase. One notable change is the disruption of an interaction between subdomain 1. 1 and domain 4. This interaction prevents the free σ70 factor from binding DNA. In essence, negatively charged subdomain 1.1 acts as a DNA mimic, which competes with promoter DNA for the binding site on domain 4.

    Darst and coworkers have also determined the crystal structure for the Taq RNA polymerase holoenzyme without bound DNA and Shigeyuki Yokoyama and coworkers have done the same for the Thermus thermopbilus RNA polymerase holoenzyme. Thus, a considerable amount of structural information is now available for the core enzyme, the holoenzyme, and the holoenzyme · fork-junction DNA complex. Based on comparisons of these structures, it appears that the bulk of the enzyme consist......

The core RNA polymerase

Posted by star on 2018-10-18 18:52:22

    Seth Darst and coworkers reported the high-resolution crystal structure for the core RNA polymerase from the bacteria T aquaticus (Taq). The protein has a total of five subunits that are present in the stoichiometry α2 ββ'ω. Each subunit is homologous with its E. coli counterpart. The ω subunit, which is not always present in isolated E. coli core RNA polymerase, may help to assemble the core RNA polymerase but is not required for RNA synthesis. The structural design of the Taq core RNA polymerase, along with the organization of its subunits.

    As evident from the orientation presented, the core polymerase resembles a crab claw. One pincer is almost entirely β subunit and the other almost entirely β' subunit. An internal groove or channel with many internal structural features runs along the full length of the core polymerase between the pincers. The channel is sufficiently wide to allow double-stranded DNA to fit inside. The core RNA polymerase is about 15 nm long (from the tips of the claws to the back) and 11 nm wide.

    Other noteworthy features of the Taq core RNA polymerase include the following:

1. The N-terminal domain (NTD) of one α subunit forms significant contacts with the corresponding domain in the other α subunit allowing the two α subunits to form a dimer. The two NTDs also bind the β-and β'-subunits. However the arrangement is not symmetrical because the NTD of one α subunit, α1 binds the β subunit while the NTD of the other α subunit, α2 binds the β' subunit. No residue in either α subunit has access to the channel of the core RNA polymerase where catalysis occure.

2. The β-and β'-subunits binds , which account for about 60% of the core RNA polymerases mass, interact extensively with each other. The catalytic site is formed by one such interaction a......

σ70-RNA polymerase combines with promoter DNA

Posted by star on 2018-10-16 19:20:58

    Interactions between σ70-RNA polymerase and promoter DNA has been examined by a technique called footprinting (FIGuRE 13. 11). A particular piece of double-stranded DNA, labeled in one strand at its 5'-termmus with 32p (or in its 3'-terminus with a labeled nucleotide), is mixed with RNA polymerase holoenzyme (or some other protein of interest that binds to DNA). Then DNase is added, but so briefly that, on average, each DNA molecule receives no more than one single-strand break. (This brief exposure to DNase is in marked contrast to the long exposure used in the DNase protection method.) Nicking occurs at all positions except those protected by σ70-RNA polymerase. The DNA is then isolated and denatured. The radioactively labeled DNA consists of a set of molecules with sizes that are determined by the nick positions in relation to the labeled 5'-end. If the DNA contains n base pairs and RNA polymerase is not added, n sizes of DNA fragments will be present. However, if RNA polymerase binds to x base pairs and thereby prevents access of the DNA to the nuclease, only n-x different sizes of DNA fragments will be represented. These fragments are separated by gel electrophoresis.

    Two DNA samples are compared, one without RNA polymerase (to obtain the positions of the n bands), and one with RNA polymerase (to determine the positions of the missing bands). The results of a DNase footprinting experiment in which a modified lactose promoter designated lac UV5 was examined. The lac UV5 promoter has the same sequence as the normal lactose promoter except that two bases in the -10 box have been changed so that the lac UV5 -10 box now has a sequence that matches the consensus sequence, which makes it a stronger promoter.

    RNA polymerase holoenzyme need not make direct contact with a base to protect it from DNase because the large DNase molecule cannot pass throug......

The σ54-RNA polymerase

Posted by star on 2018-10-16 19:03:08

    The σ54-RNA polymerase holoenzyme binds to promoters that have the two consensus sequences. However, unlike other RNA polymerase holoenzymes, it cannot activate transcription without assistance of an activator protein. The activation process, which is shown schematically, begins with the binding of activator protein to a site on the bacterial chromosome called an enhancer. which is usually 100 bp or more upstream from the σ54 promoter. Interaction between the activator proteins and σ54-holoenzyme is possible because the DNA segment between the two protein-binding sites bends to form a loop.

    Although the nucleotide sequence between the two protein binding sites may produce DNA bending, an additional protein such as the integration host factor (IHF) is sometimes required to assist the bending. Activator proteins are themselves subject to regulation by the binding of a small effector molecule or, more commonly, by the addition of a phosphate group to a specific site on the activator protein. Activation induces an ATPase activity within the activator protein that is essential for unwinding DNA in the promoter region so that transcription can begin.

Bacteria have two sigma factor families

Posted by star on 2018-10-15 00:37:21

    E. coli has seven different σ factors, which were initially distinguished based on their molecular masses and as described for σ70, the names given included the molecular mass as a superscript.Two other nomenclature systems are also used. In the first, each σ factor is indicated by the Greek letter σ followed by a superscript letter. For example, σ70 is called σD in this system. In the second system, each σ factor is named after the gene that codes for it. For example, σ70 becomes RpoD. Each kind of σ factor combines with core polymerase to form a holoenzyme that recognizes a unique set of promoter DNA sequences. The properties and functions of E. coli sigma factors are summarized. All σ proteins bind to RNA polymerase core enzymes and to DNA, usually but not always recognizing two separate DNA sites. Many possibly all σ factors, also are targets of accessory ligands that regulate their activity.

    Based on sequence homologies, E. coli factors can be divided in two major families, the σ70 family and the σ54N family). The σ54 factor is the only member of the σ54 family. All other E. coli sigma factors belong to the σ70 family. E. coli σ factors compete with one another for the core RNA polymerase. In the absence of additional regulatory factors, the rate of transcription depends solely on promoter strength and the concentration of specific holoenzymes. The RNA polymerase holoenzyme that contains σ70 transcribes genes that code for proteins required for normal exponential growth, which account for most of the bacteria's genes. Therefore, σ70 is an essential protein. RNA polymerase holoenzymes containing one of the other σ factors recognize specific sets of genes tha......

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