Transcription of RNA

Reverse transcriptase (Section 28 13) Enzyme that catalyzes the transcription of RNA to DNA Ribozyme (Section 28 11) A polynucleotide that has catalytic activity... 

Reverse transcriptase (Section 28.13) Enzyme that catalyzes the transcription of RNA to DNA. 

The presence of RNA in the cytoplasm had been linked to protein synthesis by experiments done in the early 1940s. After the discovery of the double helix, the concept followed quickly that DNA was the master "blueprint" from which secondary blueprints or transcripts of RNA could be copied. The RNA copies, later identified as messenger RNA (mRNA), provided the genetic information for specifying protein sequence. The flow of information from DNA to RNA to proteins could be symbolized as in Eq. 26-1. 

Nascent RNA. The initial transcripts of RNA, before any modification or processing. 

Nuclease-free dd O T4 DNA ligase (Promega) 0.317 Weiss U pL RNasin (Promega) 1 U pL-1 Modified, purified transcript of RNA HPLC-purified 10-mer-PEG substrate 20-mer bridging oligonucleotide... 

All classes of RNA transcripts must be processed into mature species. The reactions include several types Nucleolytic cleavage, as in the separation of the mature rRNA species from the primary transcript of RNA polymerase I action Chain extension (non-template-directed), as in the synthesis or regeneration of the common CCA sequence at the 3 end of transfer RNAs or of PolyA at the 3 end of mRNAs and Nucleotide modification, for example, the synthesis of methylated nucleotides in tRNA or rRNA. These reactions are a feature of both prokaryotic and eukaryotic gene expression, and the biological consequences are diverse. For example, modified nucleotides can affect the way in which a tRNA recognizes different codons. 

Hypotheticoi scheme for cloning cDNA frogments from reverse transcripts of RNA into M13 vectors for sequencing... 

HTV (the AIDS virus) is shown here attacking a T-4 lymphocyte. HIV is an RNA virus whose genetic material must be translated to DNA before inserting itself into the host cell s DNA. Several of the anti-AIDS drugs are directed toward stopping this reverse transcription of RNA to DNA. (Magnification 1000X)... 

Catalyze transcription of RNA nucleotide sequence into complementary DNA (cDNA). 

The RB gene encodes a transcriptional co-repressor which forms a complex with the cell-cycle-regulating transcription factor E2F (members of the E2F family of transcriptional activators control the genes involved in the Gj/S transition and in DNA replication 20 see Chapter 12). The RB—E2F transcriptional complex contains additional factors, such as the histone deacetylase, HDACl, which facilitates access of the transcriptional complex to E2F-responsive gene promoters, such as the cycKn E promoter. pRB and HDACl act in concert and repress the cyclin E promoter. i Hence the retinoblastoma protein (pRB) is a tumour-suppressor that represses gene expression in concert with a histone deacetylase, by modulating the architecture of chromatin (Fig. 15.4). The pRB- E2F transcriptional complex also represses transcription of RNA polymerase III22 (see also Chapter 9). 

Reverse transcription of RNA is sometimes used, e.g., for clone selection by colony hybridization (Verbeek and Tijssen, 1988). Although both avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptase can be used, we prefer the AMV enzyme if there are no strong secondary structures in the RNA (MMLV enzyme can be used at higher temperatures, even up to 55°C). The optimal pH range for the AMV enzyme is very narrow (should be 8.3) whereas the MMLV enzyme is active in a range of 7.6-8.3. Superscript reverse transcriptase from BRL is a cloned MMLV enzyme from which the RNase H activity has been deleted. 

Biological information circulates universally by means of three processes replication of DNA, transcription of DNA into RNA, and translation of RNA into protein. In addition, viruses with an RNA genome possess an enzyme that catalyzes one of two processes that are not carried out by cells rephcation of RNA and reverse transcription of RNA into DNA. These five processes are shown schematically in Figure 10.1. 

The primary structure of a protein is its sequence of amino acids, translated from the sequence of three-letter nucleotide codons in mRNA that itself is copied (transcribed) from DNA. The transcription of RNA, its posttranscriptional editing, processing, and transport from the nucleus to the site of translation is a huge subject, beyond the scope of this article. Interested readers are advised to consult other review articles, for example Bag (1991), Kozak (1991), and Ross (1989). 

Various methods are available for detecting and quantifying gene-expression levels, including northern blots, differential display, polymerase chain reaction after reverse transcription of RNA, and serial analysis of gene expression. These techniques are used primarily to measure the expression levels of specific genes or to screen for significant differences in mRNA abundance. 

The TATA binding protein (TBP) is a universal eukaryotic transcriptional initiation protein that is required for the transcription of RNA pol I-dependent (rRNA), RNA pol II-dependent (mRNA and snRNA), and RNA pol Ill-dependent (tRNA and 55 rRNA) genes. Auxiliary proteins called TBP-associated factors (TAFs) provide promoter-selective properties to TBP and facilitate transcriptional regulation. 

The least well defined step in eukaryotic transcription is termination, which follows the various steps of processing, discussed in Section D. The final 3 end of processed transcripts of RNA pol II action in mammals is marked by the sequence AAUAAA, which is found about 10-30 nucleotides upstream from the end. This is usually followed by a polyU or GU-rich sequence. In yeast the termination and polyadenylation signals are less clear. The initial transcript almost always continues beyond the AAUAAA signal, sometimes for hundreds of nucleotides. ITowever, the excess RNA is rapidly degraded by a large complex of... 

No. The drug acts at the level of the nucleus to alter transcription of RNA. It thus may take up to 1 to 3 months to be effective. 

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