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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.

Composition of bacterial RNA polymerase

Posted by star on 2018-10-09 19:30:24

    Several different groups detected bacterial RNA polymerases at about the same time. Purification and characterization studies revealed that the fully active form of E. coli RNA polymerase, the RNA polymerase holoenzyme, is a large multisubunit protein (molecular mass-459 kDa) that has six subunits. RNA polymerase holoenzymes from other bacteria have the same multisubunit structure and the sequence of each of the subunits appears to have been conserved during evolution.

    RNA polymerase activity is usually assayed using a reaction mixture that contains RNA polymerase (which may be in a crude extract), DNA, Mg 2+ , three nonradioactive NTPs, and one radioactive NTP labeled either in the base with 3H or 14C or in the phosphate attached to the ribose with. 32P. The reaction is stopped by adding trichloroacetic acid which causes the newly formed RNA but not the nucleoside triphosphate precursors to become insoluble, and the precipitate is collected by filtration or centrifugation. The amount of radioactivity in the precipitate is proportional to the amount of RNA synthesized.

    The large size of the bacterial holoenzyme compared to typical enzymes raises the question of whether some complex feature of the polymerization reaction requires such a large enzyme. In fact, E. coli bacteriophage T7 RNA polymerase, which has only one polypeptide subunit with a molecular mass 99 kDa and a tertiary structure (FGi-u T. ) Clearly, the polymerization reaction itself does not require a huge multisubunit bacterial enzyme.

    Clues to understanding reason for the size differences between the E. coli and the T7 phage enzymes come from attempting to use the T7 phage enzyme to transcribe E. coli DNA and studying the gene organization of the T7 phage. First, the T7 phage RNA polymerase can transcribe at best a small fraction of the E. coli genes. Second. T7 phage genes are arranged in only a few ......

Introduction to the bacterial RNA polymerase

Posted by star on 2018-10-09 19:25:44

    RNA polymerase catalyzes nucleoside monophosphate group transfer from a nucleoside triphosphate (NTP) to the 3'-end of the growing RNA chain (or the first nucleoside triphosphate) so that chain growth proceeds in a 5'→3'direction. The essential chemical characteristics of RNA synthesis are as follows:

    1. Phosphodiester bond formation takes place as the result of a nucleophilic attack of the 3'-hydroxyl group on the growing chain (or first nucleoside triphosphate) on the α phosphoryl group of the incoming NTP. This is the same type of reaction that takes place during DNA synthesis. Also, as with DNA synthesis, pyrophosphate hydrolysis drives the reaction to completion.

    2. The DNA template sequence determines the RNA sequence. Each base added to the growing 3'-end of the RNA chain is chosen by its ability to pair with a complementary base in the template strand. Thus, the bases C, T, G, and A in a DNA strand cause G, A, C, and U, respectively, to appear in the newly synthesized RNA molecule.

    3. All four ribonucleoside triphosphates (adenosine 5'-triphos-phate [ATP], guanosine 5'-triphosphate [GTP], cytidine 5'-triphosphate [CTP], and uridine 5'-triphosphate [UTP]) are required for RNA synthesis. When a single nucleotide is omitted RNA synthesis stops at the point where that nucleotide must be added.

    4. The RNA chain grows in the 5'→3'direction that is, nucleotides are added only to the 3'-OH end of the growing chain. This direction of chain growth is the same as that in DNA synthesis.

    5. RNA polymerases, in contrast with DNA polymerases, are able to initiate chain growth so that no primer is needed.

    6. 0nly ribonucleoside 5'-triphosphates participate in RNA synthesis. The first base to be laid down in the initiation event is a triphosphate. Its 3'-OH group is the point of......

Study finds new targets for triple-negative breast cancer treatment

Posted by star on 2018-10-08 18:43:12

    A new study analyzes the transcriptome characteristics of triple-negative breast cancer (TNBC) patients, and identifies and confirms the novel inhibitor Wwox protein of JAK2/STAT3 signaling in breast cancer, elucidating abnormal attenuation of Wwox and abnormal activation of JAK2/STAT3 Such a negative correlation is an important cause of the metastasis of highly malignant TNBC. The study of Loss of Wwox drives metastasis in triple-negative breast cancer by JAK2/STAT3 axis is published online in Nature-Communication.

    In recent years, the incidence of breast cancer in China has continued to rise. TNBC is a type with a high degree of malignancy. Its invasiveness is extremely strong, the risk of distant metastasis is large, and the prognosis is extremely poor. At present, its treatment is relatively simple, mainly chemotherapy, and it is easy to relapse and metastasize. Endocrine therapy and molecular targeted therapy are ineffective against TNBC compared to other types of breast cancer.

    The researchers screened transcriptomic analysis of TNBC cells and normal breast cells to obtain abnormal expression of multiple signals in the IL6/JAK2/STAT3 pathway, demonstrating the high abnormal expression of IL6 in TNBC cells and the persistence of JAK2/STAT3 activation. Activation may be an important factor in the distal metastasis of TNBC. The study also found that SOCS3, the main inhibitor of JAK2/STAT3, was not inhibited in TNBC; the newly discovered fragile site gene Wwox persisted in the IL6/JAK2/STAT3 signaling pathway in breast cancer. Specific inhibition occurs during activation. The researchers demonstrated the inhibitory effect of Wwox on sustained phosphorylation of STAT3 signaling and sustained activa......

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