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The cellular processes of DNA replication and transcription involve DNA and RNA synthesis, respectively. DNA synthesis uses dNTPs as substrates, while RNA synthesis uses rNTPs as substrates. [2] NTPs cannot be converted directly to dNTPs. DNA contains four different nitrogenous bases: adenine, guanine, cytosine and thymine.
The usage of RNA primers during DNA replication is an example of a correct incorporation of rNTPs during the process. Although, overly long RNA primers can decrease the effectiveness of T7 DNA polymerase in incorporating dNTP into the growing strand and weaken the binding between T7 and the template DNA strand.
The RNA replication process is a four-step mechanism: Nucleoside triphosphate (NTP) binding – initially, the RdRp presents with a vacant active site in which an NTP binds, complementary to the corresponding nucleotide on the template strand. Correct NTP binding causes the RdRp to undergo a conformational change. [11]
Transcription in the archaea domain is similar to transcription in eukaryotes. [25] Transcription begins with matching of NTPs to the first and second in the DNA sequence. This, like most of the remainder of transcription, is an energy-dependent process, consuming adenosine triphosphate (ATP) or other NTP.
RNAP can initiate transcription at specific DNA sequences known as promoters. It then produces an RNA chain, which is complementary to the template DNA strand. The process of adding nucleotides to the RNA strand is known as elongation; in eukaryotes, RNAP can build chains as long as 2.4 million nucleotides (the full length of the dystrophin gene).
This process is called promoter escape, and is another step at which regulatory elements can act to accelerate or slow the transcription process. Similarly, protein and nucleic acid factors can associate with the elongation complex and modulate the rate at which the polymerase moves along the DNA template.
In eukaryote cells, RNA polymerase III (also called Pol III) is a protein that transcribes DNA to synthesize 5S ribosomal RNA, tRNA, and other small RNAs. The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose expression is required in all cell types and most environmental conditions.
Pre-labelling → involves the use of 32 P: cells are grown in 32 P containing medium, thus allowing the incorporation of [α-32 P]NTPs during transcription by T7 RNA polymerase. The modified RNA is then extracted, and each RNA species is isolated and subsequently digested by T2 RNase.