DNA template strand

Nielsen P. E., Egholm M., Buchardt O. Sequence-specific transcription arrest by peptide nucleic acid bound to the DNA template strand. Gene 1994 149 139-145. 

Relationship of RNA transcript to DNA RNA is antiparallel and complementary to DNA template strand RNA is identical (except U substitutes for T) to DNA coding strand ... 

Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. Strands of RNA and DNA may seem quite similar at first glance, differing only in that RNA has a hydroxyl group at the 2 position of the al-dopentose and uracil instead of thymine. However, unlike DNA, most RNAs carry out their functions as single strands, strands that fold back on themselves and have the potential for much greater structural diversity than DNA (Chapter 8). RNA is thus suited to a variety of cellular functions. 

The discovery of DNA polymerase and its dependence on a DNA template led to the search for enzymes which could make an RNA molecule complementary to the DNA. RNA synthesis does not require a primer strand it does, however, require a specific initiation signal on the DNA template strand to allow binding and initiation. As the RNA strand is synthesised it forms a temporary helix with the template DNA, but when complete the mRNA breaks off at the stop site on DNA. Once released from DNA, some of the RNA is processed further, for the specific structures of rRNA and tRNA. 

Once the polymerase binds to the promoter and strand separation occurs, initiation usually proceeds rapidly (1 -2 s). The first, or initiating, NTP, which is usually ATP or GTP, binds to the enzyme. The binding is directed by the complementary base in the DNA template strand at the start site. A second NTP binds, and initiation occurs on formation of the first phosphodiester bond by a reaction involving the 3 -hydroxyl group of the initiating NTP with the inner phosphorus atom of the second NTP. Inorganic pyrophosphate derived from the second NTP is a product of the reaction. This process is illustrated in figure 28.10. 

As the polymerase traverses the DNA, it must continually cause a melting or strand separation of the DNA so that a single DNA template strand is available at the active site of the enzyme. During elongation, one base pair re-forms behind the active site for every base pair opened in front of it. The short transient RNA-DNA hybrid duplex that forms between the newly synthesized RNA and the unpaired region of the DNA helps to hold the RNA to the elongating complex. 

RNA is synthesized in the 5 - 3 direction by the formation of 3 -5 -phosphodiester linkages between four ribonucleoside triphosphate substrates, analogous to the process of DNA synthesis. The sequence of bases in RNA transcripts catalyzed by DNA-depen-dent RNA polymerases is specified by the complementary sequences of the DNA template strand. 

Amplification of the DNA template is achieved by PCR (for details, see [14]). The size and amount of the PCR product should be routinely checked by agarose gel electrophoresis. An insufficient amount of product may be improved by increasing the number of PCR cycles. However, care should be taken not to overdo the cycling, because this leads to a loss of dsDNA after several PCR cycles the amount of free primers decreases dramatically so that only a fraction of the DNA template strands anneal with a primer and yield new dsDNA. The high complexity of the pool causes that ssDNA is not able to rehybridize with the complementary strand. 

The transcription reaction is performed using T7 RNA polymerase according to the general protocol of Milligan et al. (1987), using a DNA template strand that incorporates 2 -OMe modifications at each of the first two nucleotides at the 5 -end to suppress formation of n + 1 transcription products (Kao et al., 1999). The sequence of the 19-mer unlabeled tagging RNA is 5 -GCaaAAGAGAUGGUGAUGGGA-3, where Caa denotes 5-aminoallyl-C. 

Fig. 4. Details of DNA replication, (a) Primase binds to the DNA template strand (thin line) and (b) synthesizes a short RNA primer (dotted line) (c) DNA polymerase III now extends the RNA primer by synthesizing new DNA (thick line) (d) during synthesis of the lagging stand, adjacent Okazaki fragments are separated by the RNA primers (e) the RNA primers are now removed and the gaps filled with DNA by DNA polymerase I (f) generating adjacent DNA fragments that are then (g) joined by DNA ligase.

Fig. 2. Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can base-pair with the next base of the DNA template strand. In the diagram, the incoming nucleotide is rUTP to base-pair with the A residue of the template DNA. A 3 5 phosphodiester bond is formed, extending the RNA chain by one nucleotide, and pyrophosphate is released. Overall the RNA molecule grows in a 5 to 3 direction.

Fig. 3. A transcription bubble. The DNA double helix Is unwound and RNA polymerase then synthesizes an RNA copy of the DNA template strand. The nascent RNA transiently forms an RNA-DNA hybrid helix but then peels away from the DNA which Is subsequently rewound into a helix once more.

Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves along the template, locally unzipping the DNA double helix. This allows a transient base pairing between the incoming nucleotide and newly-synthesized RNA and the DNA template strand. As it is made, the RNA transcript forms secondary structure... 

The mRNA sequence is the complement of the DNA template strand, which is the complement of the DNA coding strand. Thus, the mRNA sequence is a copy of the DNA coding strand, with T replaced by U. 

RNA polymerase, like the DNA polymerases described earlier, takes instructions from a DNA template. The earliest evidence was the finding that the base composition of newly synthesized RNA is the complement of that of the DNA template strand, as exemplified by the RNA synthesized from a template of single-stranded ( ) XI74 DNA (Table 5 3). Hybridization experiments also revealed that RNA synthesized by RNA polymerase is complementary to its DNA template. In these experiments, DNA is melted and allowed to reassociate in the presence of mRNA. RNA-DNA hybrids will form if the RNA and DNA have complementary sequences. The strongest evidence for the fidelity of transcription came from base-sequence studies showing that the RNA sequence is the precise complement of the DNA template sequence (Figure 5.26). 

Figure 5.26. Complementarity between mRNA and DNA. The base sequence of mRNA (red) is the complement of that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA containing the genes for five enzymes that catalyze the synthesis of tryptophan. The other strand of DNA (black) is called the coding strand because it has the same sequence as the RNA transcript except for thymine (T) in place of uracil (U).

Encoded sequences, (a) Write the sequence of the mRNA molecule synthesized from a DNA template strand having the sequence... 

Although RNA polymerase can search for promoter sites when bound to double-helical DNA, a segment of the helix must be unwound before synthesis can begin. A region of duplex DNA must be unpaired so that nucleotides on one of its strands become accessible for base-pairing with incoming ribonucleoside triphosphates. The DNA template strand selects the correct ribonucleoside triphosphate by forming a Watson-Crick base pair with it (Section 5.2.1). as in DNA synthesis. 

How does this combination hairpin-oligo(U) structure terminate transcription First, it seems likely that RNA polymerase pauses immediately after it has synthesized a stretch of RNA that folds into a hairpin. Furthermore, the RNA-DNA hybrid helix produced after the hairpin is unstable because its rU-dA base pairs are the weakest of the four kinds. Hence, the pause in transcription caused by the hairpin permits the weakly bound nascent RNA to dissociate from the DNA template and then from the enzyme. The solitary DNA template strand rejoins its partner to re-form the DNA duplex, and the transcription bubble closes. 

What is the sequence of the DNA coding strand Of the DNA template strand ... 

A. Replication occurs during the S phase of the cell cycle. Bubbles on the parental DNA serve as points of origin from which replication proceeds in both directions simultaneously. (Replication is bidirectional.) DNA template strands are always copied in a 3 to 5 direction. New strands are synthesized 5 to 3. The daughter molecules each contain one parental strand and one newly synthesized strand. (Replication is semiconservative.)... 

A. Transcription is catalyzed by RNA polymerase II, which binds to promoter regions, including a TATA box. The DNA template strand is not covalently bound to histones. The primary transcript (hnRNA) is capped at the 5 end and polyadenylated at the 3 end introns are removed by splicing to form mRNA. 

The sequence of bases in an RNA molecule is determined by the base sequence of the DNA template strand. Each base added to the growing end of an RNA chain is chosen by base pairing with the appropriate base in the template strand thus, the bases C, T, G, and A in a DNA strand cause incorporation of G, A, C, and U, respectively, in the newly synthesized RNA molecule. The RNA is complementary to the template DNA strand, which is called the coding (sense) strand or template strand. 

RNA polymerase, like the DNA polymerases described earlier, takes instructions from a DNA template. The earliest evidence was the finding that the composition of newly synthesized RN A is the complement of that of the DNA template strand, as exemplified by the RNA synthesized from a template of single-stranded DNA from the ct)X174 virus (Table 4.3). 

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