The question of whether the template strand is always read 3′ to 5′ during transcription, where a segment of DNA is copied into RNA, can be answered directly: yes, it is. The directionality of reading the template strand is a fixed rule dictated by the fundamental mechanics of the enzyme responsible for creating the RNA molecule. This universal directionality is a consequence of the chemical structure of nucleic acids and the limitations of the polymerase enzyme. Understanding this requires examining DNA polarity and the enzyme’s function that enforces this unidirectional reading.
Understanding DNA Polarity
The concept of “directionality” in a DNA strand is rooted in the chemical structure of its building blocks, the nucleotides. Each nucleotide is composed of a phosphate group, a deoxyribose sugar, and a nitrogenous base. The 5′ (five-prime) and 3′ (three-prime) designations refer to specific carbon atoms on this sugar.
The 5′ end is defined by the free phosphate group attached to the 5th carbon of the sugar molecule. Conversely, the 3′ end is defined by a free hydroxyl group (-OH) attached to the 3rd carbon. When nucleotides link together, the phosphate group of one forms a phosphodiester bond with the 3′ hydroxyl group of the next, creating a chain with a distinct chemical orientation. This polarity means that the two strands of the DNA double helix run antiparallel, with the 5′ end of one strand aligning with the 3′ end of the opposite strand.
The RNA Polymerase Rule
The enzyme responsible for transcription, RNA polymerase, operates under a strict constraint that governs all nucleic acid synthesis. This enzyme can only synthesize a new strand of RNA by adding new nucleotides to the existing 3′ end of the growing chain. Therefore, the RNA molecule is always constructed in the 5′ to 3′ direction.
This requirement for 5′ to 3′ synthesis is due to the energy-dependent mechanism used by the polymerase, which relies on nucleoside triphosphates to provide the energy for phosphodiester bond formation. The enzyme physically moves along the DNA template, adding one complementary ribonucleotide at a time to the free hydroxyl group at the 3′ terminus of the nascent RNA. This inherent limitation of the polymerase enzyme determines the direction in which the DNA template must be read.
How the Template Strand is Read
The RNA polymerase’s rule of synthesizing RNA in the 5′ to 3′ direction directly necessitates that the DNA template strand be read in the opposite, antiparallel direction, which is 3′ to 5′. As the enzyme moves, it separates the two DNA strands, creating a transcription bubble where one strand is exposed to be used as the template. The enzyme physically travels along this template strand from the 3′ end toward the 5′ end.
During this movement, the enzyme reads the sequence of bases on the template strand and recruits complementary RNA nucleotides to build the new RNA molecule. This specific DNA strand is known as the template strand, or antisense strand. The other DNA strand, which has a sequence identical to the resulting RNA (save for the substitution of Uracil for Thymine), is called the coding or sense strand.
Template Switching and Gene Orientation
While the reading direction of any template strand is unchangeably 3′ to 5′, the actual physical strand of the double helix that serves as the template is not fixed for the entire chromosome. Genes are not all arranged on the same strand; rather, they can be oriented in either direction on the DNA. This flexibility means that for one gene, the upper physical strand might be the template, but for a neighboring gene, the lower physical strand might be used.
The orientation of a gene is determined by the location of its promoter region, which is the binding site that directs the RNA polymerase where to start transcription. The promoter dictates which of the two strands will be the template and in which direction the polymerase will move, which will always be 3′ to 5′ along the chosen template strand. This genomic organization, where genes can be transcribed from either strand, is referred to as template switching. This process does not violate the fundamental 3′ to 5′ reading rule; it simply changes the physical strand being read.