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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......

The DNase protection rnethod provides information about promrther DNA

Posted by star on 2018-10-11 23:35:41

    RNA polymerase holoenzyme is sufficiently large to contact many deoxyribonucleotides within the promoter simultaneously. An estimate of the size of the region of the DNA where contact is made is obtained by selectively degrading adjacent DNA bases with DNase a procedure known as the DNase protection method. RNA polymerase holoenzyme is bound to DNA and then a DNA endonuclease is added to the mixture. The endonuclease degrades most of the DNA to mono-and dinucleotides but leaves untouched DNA segments in close contact with RNA polymerase; protected segments vary in size from 41 to 44 base pairs (bp). If RNA polymerase holoenzyme were to be added to the total DNA complement of E. coil and then DNase were added, the protected promoter fragments would consist of about a thousand different DNA segments, each derived from a particular gene or set of adjacent genes. To study details of the DNA-enzyme binding process, it is obviously desirable to examine a single binding sequence. This can be accomplished by using a cloned gene in a protection experiment.

    David Pribnow used the DNase protection method to obtain DNA fragments containing promoters from genes that had been protected by RNA polymerase holoenzyme. These fragments were sequenced as were RNA molecules synthesized from each gene in vitro. The 5'-terminus of the RNA molecule revealed the initiation start site on the complementary DNA template strand. Pribnow recognized an important common feature in the protected DNA fragments. A six base pair sequence centered about 10 bp before (upstream from) the transcription start site is conserved. This hexamer is now known as the-10-box for its location or the Pribnow box for its discoverer. Examination of 300 E. coli promoters has shown that the frequency of occurrence of bases in-10 boxes is as follows (the subscript is the frequency):


How to achieve accurate and efficient transcription

Posted by star on 2018-10-10 19:17:24

    In contrast to the core polymerase (α2ββ'), the RNA polymerase holoenzyme (α2ββ'σ) can use intact DNA as a template for RNA synthesis. Transcription begins when the holoenzyme recognizes and binds to specific initiation signals called promoters at the beginning of a transcription unit. The existence of promoters was first demonstrated by isolation of a particular class of mutations in E. coli that prevent cells from synthesizing enzymes required for lactose metabolism. These mutations, termed promoter mutations, not only result in a lack of gene activity but also cannot be complemented because they are cis-dominant.

    Sigma factor is essential for promoter DNA recognition but does not bind to promoter DNA on its own. The major E. coli σ factor, called σ70 because its molecular mass is about 70 kDa, helps core polymerase to bind to promoters in genes that code for housekeeping enzymes (enzymes required for essential metabolic steps in the cell)and destabilizes nonspecific interactions between RNA polymerase and DNA. Other bacterial species have σ70 homologs that perform the same function. The fact that core RNA polymerase can transcribe nicked DNA but not intact DNA suggested that σ70 might function by introducing transient nicks into DNA. Despite extensive efforts to observe such a nicking activity, none has ever been observed. For in-stance, σ70 does not alter the linking number of supercoiled DNA.

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