Transcription-translation

In all types of mammalian cells, the molecular mechanism underlying circadian clock function is based upon interconnected transcription/translation feedback loops [2] (Fig. 2). Two proteins that function as transcriptional... 

Na+/Ca2+ Exchangers. Figure 3 Potential steps in the pharmacological regulation of NCX isoform expression and activity. This scheme reproduces the potential levels at which drugs can interfere with the transduction, transcription, translation, and activity of NCX. 

Both the heat and cold shock response are universal and have been studied extensively. The major heat shock proteins (HSPs) are highly conserved. They are involved in the homeostatic adaptation of cells to harsh environmental conditions. Some act as molecular chaperones for protein folding, while others are involved in the processing of denatured polypeptides whose accumulation would be deleterious. The cold shock results in the transient induction of cold shock proteins (CSPs), which include a family of small acidic proteins carrying the cold shock domain. The CSPs appear to be involved in various cellular functions such as transcription, translation and DNA recombination. 

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

Ozone attacks the rings of PAHs rather indiscriminately with fission of the rings to prodnce aldehyde gronps. There has been concern, however, since the products may be more harmful than their precursors. In the studies that are used as illustration, in vitro gap junctional intracellular communication (GJIC) was used to assess adverse alteration on the expression of genes at the transcription, translational, or posttranslational level ... 

The protein synthesis machinery reads the RNA template starting from the 5 end (the end made first) and makes proteins beginning with the amino terminus. These directionalities are set up so that in prokaryotes, protein synthesis can begin even before the RNA synthesis is complete. Simultaneous transcription-translation can t happen in eukaryotic cells because the nuclear membrane separates the ribosome from the nucleus. 

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

Regulation of neuropeptide expression is exerted at several levels. Control of neuropeptide function is mediated by factors controlling rates of prepropeptide gene transcription, translation, peptide degradation and secretion (Fig. 18-11). On the scale of seconds to minutes, peptide secretion is not always coupled lock-step with classical transmitter release (example above). Peptides are inactivated by diffusion and by proteolysis, so it would be expected that inhibition of specific extracellular proteases... 

Manipulation of one enzymatic step in a system can have wide reaching consequences because of the interplay between metabolite levels and a wide range of regulatory circuits. These circuits can operate at the level of transcription, translation, post-translational modification, or through allosteric and competitive influences on the kinetic properties of enzymes. 

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

RTS rapid translation system, a transcription/translation system used for polypeptide production in vitro... 

The Lester and Dougherty labs, which have collaborated to extend the suppression mutagenesis technique to Xenopus oocytes with remarkable success [30, 31], began with a suppressor tRNA ( MN3 ) designed for in vivo use and demonstrated that it functioned more effectively in the oocyte system than a yeast tRNA -derived suppressor tRNA. They have since developed an alternative suppressor based on tRNA " from Tetrahymena thermophila that has proven to be considerably more versatile, efficient and accurate in the oocyte system [32], as well as showing good suppression efficiency in E. coli transcription-translation reactions [33]. 

The ability of both suppressor tRNAs to incorporate the nonpolar amino acid valine as well as the polar noncoded homoglutamate into two proteins was tested in E. coli cell-free transcription-translation systems [35]. The proteins T4... 

When the DNA templates were added to M. luteus transcription-translation reactions containing the corresponding charged suppressor tRNAs, full-length... 

Figure 7.7. Schematic representation of gene selection by compartmentalization. Step 1 An in vitro transcription/translation reaction mixture containing a library of genes linked to a substrate for the reaction being selected is dispersed to form a water-in-oil emulsion with typically one gene per aqueous compartment. Step 2 The genes are transcripted and translated within their compartments. Step 3 Proteins (or RNAs) with enzymatic activities convert the substrate into a product that remains linked to the gene. Compartmentalization prevents the modification of genes in other compartments. Step 4 The emulsion is broken all reactions are stopped and the aqueous compartments are combined. Genes linked to the product are selectively enriched, then amplified, and either characterized (step 5) or linked to the substrate and compartmentalized for further rounds of selection (step 6). (Adapted from [39].)...

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