Template antisense strand

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

When you see a sequence written with only one strand shown, the 5 end is written on the left. Usually this sequence is also identical to that of the RNA that would be made from this piece of DNA when transcribed left to right. The DNA strand that has the same sequence (except U for T) as the RNA that is made from it is called the sense strand. The sense strand has the same sequence as the mRNA. The antisense strand serves as the template for RNA polymerase. 

RNA polymerase uses the antisense strand of DNA as a template. RNA is synthesized in the 5 to 3 direction. 

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-... 

Unlike the DNApolymerase reaction, RNApolymerases catalyze the transcription of only one of the two DNA strands. The two DNA strands are termed the sense strand and the antisense strand. It is the antisense strand that is transcribed by the RNA polymerases. Thus, the base sequence of the newly synthesized RNA strand is identical to the sense strand of the DNA template, except of conrse that U replaces T. 

F ig U re 1 3.1 A schematic view of RNA chain elongation catalyzed by an RNApolymerase. In the region being transcribed, the DNA double helix is unwound by about a turn to permit the DNAs sense strand to form a short segment of DNA-RNA hybrid double helix. That forms the transcription bubble. Note that the DNA bases in the bubble on the antisense strand are now exposed to the enzyme and are useable as a template for chain elongation. The RNApolymerase works its way down the DNA molecule until it encounters a stop signal. (Reproduced from D. Voet and J. G. Voet, Biochemistry, 3rd, edn, 2004 Donald and Judith G Voet. Reprinted with permission of John Wiley and Sons, Inc.)... 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

Each RNA polymerase transcribes only one strand, the antisense (—) strand, of a double-stranded DNA template, directed by a promoter. Synthesis occurs 5 — 3 and does not require a primer. 

During transcription of information from DNA into mRNA, the two complimentary strands of the DNA partly uncoil. The sense strand separates from the antisense strand. The antisense strand of DNA is used as a template for transcribing enzymes that assemble mRNA (transcription), which, in the process produces a copy of the sense strand. Then, mRNA migrates into the cell, where other cellular structures called ribosomes read the encoded information, its mRNA s base sequence, and in so doing, string together amino acids to form a specific protein. This process is called translation. ... 

The sequence of nucleotides within the single-stranded mRNA is assembled according to the complementary-base-pairing (Chap. 7) instructions from one of the strands of duplex DNA, which contains the gene. The DNA strand that bears the same sequence as the mRNA (except for T instead of U) is called the coding strand or sense strand. The other strand of DNA which acts as the template for transcription is called the template or antisense strand. Some textbooks do not define sense" and antisense in the way described here, and for this reason it may be preferable to use coding and template when referring to a particular strand. 

The process of DNA-dependent RNA synthesis is catalysed by RNA polymerases and requires the precursor nucleoside 5 -triphosphates (ATP, GTP, CTP and UTP, each as Mg2+ complexes) and a template (i.e. the DNA being transcribed ). The reaction proceeds in a 5 to 3 direction, that is, at the end of the synthesis there is a vacant ribose 3 -OH. The fidelity of the replication process is based on the incoming nucleotides base- pairing with the correct base on the antiparallel antisense ssDNA template that has to unwind from its complementary sense strand during the process. 

The first nucleotide (the start site) of a transcribed DNA sequence is denoted as +1 and the second one as +2 the nucleotide preceding the start site is denoted as -1. These designations refer to the coding strand of DNA. Recall that the sequence of the template strand of DNA is the complement of that of the RNA transcript (see Figure 5.26). In contrast, the coding strand of DNA has the same sequence as that of the RNA transcript except for thymine (T) in place of uracil (U). The coding strand is also known as the sense (+) strand, and the template strand as the antisense (-) strand. 

Nucleotide incorporation is determined by base pairing with the template strand of the DNA. The template is the DNA strand, also called the sense strand, that is copied by the RNA polymerase into a complementary strand of RNA called the transcript. The DNA strand that is not copied is know as the antisense strand. Note that while the RNA chain grows in a 5 to 3 direction the polymerase migrates along the sense strand in a 3 to 5 direction. Thus, the 5 to 3 ribonucleotide sequence of the RNA transcript is identical to the 5 to 3 antisense DNA strand with uracil in place of thymidine. 

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 called the coding strand, or sense strand, and the strand that gets transcribed is called the template strand, or antisense strand. Since the template strand and the coding strand are... 

The sequence of the coding ( +, sense) strand is 5 -ATGGGGAACAGCAAGAGTGGGGCCCTGTCCAAGGAG-3 and the sequence of template ( —, antisense) strand is... 

The strand that the RNA polymerase uses as a template for its RNA is called the template strand, the noncoding strand, the antisense strand, and the (-) strand. The other strand, whose sequence matches the RNA produced except for the T-U change, is called the nontemplate strand, the coding strand, the sense strand, and the (+) strand. 

Like prokaryotic RNA polymerase, each eukaryotic enzyme copies DNA from the 3 end, thus catalyzing mRNA formation in the 5 => 3 direction and synthesizing RNA complementary to the antisense DNA template strand. The reaction requires the precursor nucleotides ATP, GTP, CTP, and UTP and does not require a primer for transcription initiation. Unlike the prokaryotic bacterial polymerases, the eukaryotic RNA polymerases require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription. The five stages of eukaryotic transcription include initiation, elongation and termination, capping, polyadenylation, and splicing. 

Recognize the convention for numbering the nucleotides in the DNA template with regard to the transcription start site. Distinguish between the template (or antisense) strand and the coding (or sense) strand of the duplex DNA template. 

The sequence of nucleotides in a single-stranded mRNA is assembled according to complementary base pairing with one of the two strands of duplex DNA that contains the gene. One DNA strand thus provides the template for transcription it is called the template or antisense strand (Fig. 9-1). The other DNA strand has the same sequence as the mRNA (except with T in place of U) and is called the coding, or sense, strand. Generally, it is the coding strand of DNA that is quoted when a DNA sequence is written. 

In northern blots, mRNA is separated by size on an electrophoresis gel, then immobilized on a nylon membrane. Therefore, it is the template (antisense) strand of the probe that will hybridize to the mRNA. 

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