Transcriptional Processes

Transcription is a highly complex process because of its defined initiation and termination sites in the genome and the subsequent processing and regulation of its synthesis. The steady-state level of a protein in the cell is the balance of its rate of synthesis and degradation. The synthesis is determined primarily by the steady-state level of its mRNA. Thus, the rate of transcription often determines the level of its gene product in vivo.

As mentioned earlier,RNAsynthesis is catalyzed by the RNA polymerase in all organisms. Prokaryotes express a single RNA polymerase used for synthesis of all RNAs, while eukaryotes encode multiple RNA polymerases with dedicated functions. RNA polymerase I (Pol I) in eukaryotic cells is responsible for synthesis of ribosomal RNA, which accounts for more than 70% of total RNA in the cell. Pol III catalyzes synthesis of small RNA molecules, including transferRNAs which bring in appropriate amino acids to the ribosome for protein synthesis by using their “anti-codon” triplet bases. Pol II is responsible for synthesis of all other RNA, specifically mRNA.

RNA polymerases of all organisms are complex machines consisting of multiple subunits which alter conformation. A variety of structural analyses show the presence of a 2.5-nm-wide “channel” on the surface of all DNA polymerases which could be the path for DNA. The RNA polymerase holoenzyme binds to a promoterspecific recognition sequence upstream (5´ side of the transcribed strand) of the site of synthesis initiation. While the RNA polymerase is normally present as a closed complex with nonspecific DNA, in which DNA base pairs are not broken, a significant conformational change produces the open complex whenRNAthe enzyme binds the promoter, unwinds the DNA duplex, and is poised to initiate RNA synthesis.

As in the replication process, initiation is the first stage in transcription and denotes the formation of first phosphodiester bond. Unlike in the case of DNA synthesis, RNA chains are initiated de novo without the need of a primer. However, when a primer oligonucleotide is present, RNA polymerases can also extend the primer as dictated by the template strand. A purine nucleotide invariably starts the RNA chains in both prokaryotes and eukaryotes, and the overall rate of chain growth is about 40 nucleotides per second at 37◦C in E. coli. This rate is much slower than that for DNA chain elongation (∼800 base pairs per second at 37◦ for the E. coli genome).

RNA synthesis is not monotonic, andRNApolymerases can move backward like DNA polymerases do for their editing function in which an incorrectly inserted deoxynucleotide is removed by 3´ exonuclease activity. RNA polymerases stall, back track, and then cleave off multiple newly inserted nucleotides at the 3´ terminus. Subsequently, polymerases move forward along the DNA template and resynthesize the cleaved region. Based on the segment of DNA covered by an RNA polymerase as analyzed by DNA footprinting, it has been proposed that the enzyme alternatively compresses and extends in its binding to the DNA template and acts like an inchworm in its transit.

RNA polymerases of both prokaryotes and eukaryotes function as complexes consisting of a number of subunits. The E. coli RNA polymerase enzyme with a total molecular mass of about 465 kD contains two α-subunits, one β- and one β´-subunit each, and a σ-subunit which provides promoter specificity. During chain elongation, a ternary complex of macromolecules among DNA template, RNA polymerase, and nascent RNA is maintained in which most of the nascent RNA molecule is present in a singlestranded unpaired form. The stability of the complex is maintained by about nine base pairs between RNAand the transcribed (noncoding) DNA strand at the growing point.

While DNA replication warrants permanent unwinding of the parental duplex DNA, asymmetric copying of only one strand by RNA polymerase requires localized strand separation which is induced by the polymerase itself, resulting in a transcription bubble. During chain elongation, this bubble moves along the DNA duplex. Initiation of RNA synthesis is enhanced in an in vitro reaction with supercoiled duplex circular DNA template in which base pairs are destabilized due to torsional stress. Unwinding of the helix at the transcription site causes overwinding (positive supercoiling) of the template DNA ahead of the transcription bubble and underwinding (negative supercoiling) behind the bubble.