Transcription primer strand

Unlike what happens in DNA replication, where both strands are copied, only one of the two DNA strands is transcribed into mRNA. The strand that contains the gene is often called the coding strand, or primer strand, and the strand that gets transcribed is called the template strand. Because the template strand and the coding strand are complementary, and because the template strand and the transcribed RNA are also complementary, the RNA )no ecule produced during transcription is a copy of the DNA coding strand. The only difference is that the RNA molecule has a U everywhere the DNA coding strand has a T. 

Transcription (DNA), 1108-1109 coding strand in, 1108 primer strand in, 1108 promoter sites in, 1108 template strand in, 1108 Transfer RNA, 1108... 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

Using ribonucleotide 5 -phosphoro-2-methylimidazolide monomers, non-en-zymatic transcription has been demonstrated from a DNA hairpin template. The primer strand of the hairpin was substituted with rG at the 3 -end. The template strand incorporated an isoguanosine (iso-dG) residue, and the experiments demonstrated that iso-dG could act as a template for isocytidine. 

RNA polymerase makes a copy of the sense strand of the DNA using the antisense strand as a template (Fig. 5-8). The sequence of the primary transcript is the same as that of the sense strand of the DNA. RNA polymerase needs no primer—only a template. Either of the two DNA strands can serve as the template strand. Which DNA strand is used as the tern-... 

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

Primer A short length of single-stranded DNA or sometimes RNA (usually 20 bases which can be synthesized) which is complementary to a known DNA sequence so that it can bind there and serve for the initiation of DNA replication. Promoter Usually a specific region of DNA at which RNA-polymerase binds and initiates transcription. 

RNA polymerase locates genes in DNA by searching for promoter regions. The promoter is the binding site for RNA polymerase. Binding establishes where transcription begins, which strand of DNA is used as the template, and in which direction transcription proceeds. No primer is required. 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

Our discussion of RNA synthesis begins with a comparison between transcription and DNA replication (Chapter 25). Transcription resembles replication in its fundamental chemical mechanism, its polarity (direction of synthesis), and its use of a template. And like replication, transcription has initiation, elongation, and termination phases—though in the literature on transcription, initiation is further divided into discrete phases of DNA binding and initiation of RNA synthesis. Transcription differs from replication in that it does not require a primer and, generally, involves only limited segments of a DNA molecule. Additionally, within transcribed segments only one DNA strand serves as a template. 

Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters (described below). The 5 -triphos-phate group of the first residue in a nascent (newly formed) RNA molecule is not cleaved to release PPj, but instead remains intact throughout the transcription process. During the elongation phase of transcription, the growing end of the new RNA strand base-pairs temporarily with the DNA template to form a short hybrid... 

Antisense RNA. Another mechanism of control of either transcription or of plasmid replication involves small molecules of RNA that are transcribed from the opposite strand than the template strand used for mRNA synthesis.1 13 165-166b These antisense RNA molecules have at least some part of their sequence complementary to that of the mRNA and to the corresponding sequence in DNA. A well-studied example is control of the copy number of the colicin El and other plasmids of E. co/i.167-169 Two transcripts, RNAI and RNAII, are initiated upstream from the origin of replication (Fig. 28-8). RNA II is a 555-nucleotide primer of replication. It is synthesized as a longer transcript that is cut by RNase H at ori. This... 

Figure 28-8 Simplified scheme for control of replication of the ColEl type plasmid by antisense RNA. The primer for DNA synthesis is RNA II whose initial transcript extends past the replication ori. It is cut by RNase H at ori and then primes replication of the upper strand as shown in the figure. The antisense RNA is RNA I. It hinds to protein Rop whose gene location is also indicated in the figure. Rop assists RNA I and RNA II in undergoing a complementary interaction. However, both RNAs apparently maintain a folded tertiary structure, and only some segments interact. The interaction with the Rop protein evidently in some way prevents initiation of replication until the Rop concentration falls because of replication of the host cell.167,168...

The first step of this process involves PCR amplification of the target region. The use of a primer that carries a T7 promoter sequence at its 5 end enables incorporation of a transcription start site in the PCR product. This allows for in vitro transcription of the PCR product into a single-stranded RNA molecule in a subsequent step. Endonucleolytic cleavage of the RNA molecule is then performed by adding a ribonuclease. RNase A cleaves pyrimidine residues (Cytosine and Uracil) and therefore it is not... 

To make replication and transcription simple a rolling circle of DNA is envisioned which would react with coilphage DNA polymerase to produce a single strand of DNA that can be transcribed into RNA. This must include the sequence of ribosomal RNA for protein synthesis, segments of which would also have to function as DNA primers. While this appears to be simple at the theoretical level the major challenge will be in the complex interplay between replication, transcription and eventual translation into functional proteins. The key step will be to generate the correct translation machinery. As Church points out, almost all the essential genes... 

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