RNA transcript

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

FIGURE 13.15 Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SPG are useful for making RNA transcripts in vitro. SPG RNA polymerase works efficiently in vitro and recognizes its specific promoter with high specificity. 

In mammalia, seven different members encoded by distinct genes have been identified, all of which are activated by a distinct set of cytokines. Diversity in signaling is provided by variants of STAT proteins derived from either alternative splicing of RNA transcripts or proteolytic processing (e.g., STATs 1,3,4, and 5) and the ability of certain STATs to form both homodimers and heterodimers with each other. In response to inteiferon-y monomeric STAT1 dimerizes, while upon interferon-a stimulation a heterotrimeric complex consisting of STAT 1 and STAT2 with associated... 

Spike-ins are usually RNA transcripts used to calibrate measurements in a DNA microarray experiment. Each spike-in is designed to hybridize with a specific control probe on the target array. Manufacturers of commercially available microarrays typically offer companion RNA spike-ins kits . Known amounts of RNA spike-ins are mixed with the experiment sample during preparation. Subsequently the measured degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements of the sample RNA. 

Zeng F, Schultz RM. RNA transcript profiling dnring zygotic gene activation in the preimplantation mouse embryo. Dev Biol 2005 283 40-57. 

Figure 35-8. The relationship between the sequences of an RNA transcript and its gene, in which the coding and template strands are shown with their polarities. The RNA transcript with a 5 to 3 polarity is complementary to the template strand with its 3 to 5 polarity. Note that the sequence in the RNA transcript and its polarity is the same as that in the coding strand, except that the U of the transcript replaces theT of the gene.

Figure 37-2. RNA polymerase (RNAP) catalyzes the polymerization of ribonucleotides into an RNA sequence that is complementary to the template strand of the gene. The RNA transcript has the same polarity (5 to 3 ) as the coding strand but contains L) rather than T. E coli RNAP consists of a core complex of two a subunits and two p subunits (P and p ). The holoen-zyme contains the 0 subunit bound to the ajPP core assembly. The co subunit is not shown. The transcription "bubble" is an approximately 20-bp area of melted DNA, and the entire complex covers 30-75 bp, depending on the conformation of RNAP.

Figure 37-3. The transcription cycle in bacteria. Bacterial RNA transcription is described in four steps ...

Figure 37-6. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat (the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure. Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the p termination factor interacts with the paused polymerase and somehow induces chain termination.

As mentioned above, mammahan mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly(A) tail at the 3 terminal. The cap stmcmre is added to the 5 end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mELNA molecule to the cytoplasm. The S cap of the RNA transcript is required both for efficient translation initiation and protection of the S end of mRNA from attack by S —> S exonucleases. The secondary methylations of mRNA molecules, those on the 2 -hydroxy and the N of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm. 

The genetic information within the nucleotide sequence of DNA is transcribed in the nucleus into the specific nucleotide sequence of an RNA molecule. The sequence of nucleotides in the RNA transcript is complementary to the nucleotide sequence of the template strand of its gene in accordance with the base-pairing rules. Several different classes of RNA combine to direct the synthesis of proteins. 

The version 2.0 assay uses a different set of probes designed to hybridize to genotypes 1 to 6 with equal efficacy (Fig. 4). The new probe set not only enhanced the efficiency of binding to genotypic variants but also lowered the LOQ from 3.5 X 105 to 2 X 105 HCV RNA equivalents/ml (Detmer et al., 1996). The version 2.0 assay displayed almost a 600-fold dynamic range up to 1.2 X 108 RNA equivalents/ml. The LOQ was set at 2 X 105 to ensure a specificity of 95%. The assay was reproducible, with a mean CV of 14% for replicates of low-, middle-, and high-titer sera. Serial dilutions of quality level 1 RNA transcripts (Collins et al,... 

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

Propst, F Rosenberg, M. P., Iyer, A., Kaul, K and Vande Woude, G. F. (1987). c-mos proto-oncogene RNA transcripts in mouse tissues structural features, developmental regulation, and localization in specific cell types. Mol. Cell. Biol. 7 1629-1637. 

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