Transcription (biology)

About: Transcription (biology) is a research topic. Over the lifetime, 56532 publications have been published within this topic receiving 2952782 citations. The topic is also known as: genetic transcription & transcription, genetic.

In vitro RNA synthesis with SP6 RNA polymerase.

Abstract: Publisher Summary This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 RNA polymerase. The chapter describes the use of in vitro transcription systems for the synthesis of RNAs for use as substrates and hybridization probes. The chapter discusses the advantages of the technique and several associated problems. The system of SP6 RNA polymerase is used as an example throughout the chapter, but the observations are stated to be general and can be applied to any of the phage polymerase and promoter systems, including T3 and T7. The chapter discusses the in vitro transcription with bacteriophage RNA polymerase, vectors containing bacteriophage promoters, RNA synthesis reaction, and several related concepts. The optimum conditions for in vitro transcription of deoxyribonucleic acid (DNA) cloned into plasmids containing an SP6 promoter are described in the chapter. The normal product of a transcription reaction contains a triphosphate group at the 5' end of the molecule. RNA is very stable when stored as an ethanol suspension and unlike DNA it does not seem to aggregate. As a result, very reproducible samples of RNA can be taken from an ethanol suspension providing that it is vortexed prior to setting up the reaction. The chapter reviews some of the problems most commonly encountered when using in vitro transcription systems and some potential solutions.

Probing the structure of RNAs in solution

Abstract: During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors

Abstract: The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region.

Abstract: Genomic clones coding for human fibroblast collagenase were isolated. By constructing and transfecting mutants with 5' and 3' deletion mutations of the 5' control region of the gene into human or murine cells, we delimited a 32-base-pair sequence at positions -73 to -42 which is required for the induction of transcription by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The DNA element behaves as a 12-O-tetradecanoyl-phorbol-13-acetate-inducible enhancer: it mediates the stimulation of transcription to the heterologous herpes simplex virus thymidine kinase promoter and acts in a position- and orientation-independent manner. Differences in enhancer efficiency in different cell lines are interpreted to indicate differences in the activity of a trans-acting factor.