Harpers

344 / CHAPTER 37cule from the 5′ to its 3′ end continues cyclically, antiparallelto its template. The enzyme polymerizes theribonucleotides in a specific sequence dictated by thetemplate strand and interpreted by Watson-Crick basepairingrules. Pyrophosphate is released in the polymerizationreaction. This pyrophosphate (PP i ) is rapidlydegraded to 2 mol of inorganic phosphate (P i ) by ubiquitouspyrophosphatases, thereby providing irreversibilityon the overall synthetic reaction. In both prokaryotesand eukaryotes, a purine ribonucleotide is usuallythe first to be polymerized into the RNA molecule. Aswith eukaryotes, 5′ triphosphate of this first nucleotideis maintained in prokaryotic mRNA.As the elongation complex containing the coreRNA polymerase progresses along the DNA molecule,DNA unwinding must occur in order to provide accessfor the appropriate base pairing to the nucleotides ofthe coding strand. The extent of this transcription bubble(ie, DNA unwinding) is constant throughout transcriptionand has been estimated to be about 20 basepairs per polymerase molecule. Thus, it appears that thesize of the unwound DNA region is dictated by thepolymerase and is independent of the DNA sequence inthe complex. This suggests that RNA polymerase hasassociated with it an “unwindase” activity that opensthe DNA helix. The fact that the DNA double helixmust unwind and the strands part at least transientlyfor transcription implies some disruption of the nucleosomestructure of eukaryotic cells. Topoisomerase bothprecedes and follows the progressing RNAP to preventthe formation of superhelical complexes.Termination of the synthesis of the RNA moleculein bacteria is signaled by a sequence in the templatestrand of the DNA molecule—a signal that is recognizedby a termination protein, the rho (ρ) factor. Rhois an ATP-dependent RNA-stimulated helicase thatdisrupts the nascent RNA-DNA complex. After terminationof synthesis of the RNA molecule, the enzymeseparates from the DNA template and probably dissociatesto free core enzyme and free σ factor. With theassistance of another σ factor, the core enzyme thenrecognizes a promoter at which the synthesis of a newRNA molecule commences. In eukaryotic cells, terminationis less well defined. It appears to be somehowlinked both to initiation and to addition of the 3′polyA tail of mRNA and could involve destabilizationof the RNA-DNA complex at a region of A–U basepairs. More than one RNA polymerase molecule maytranscribe the same template strand of a gene simultaneously,but the process is phased and spaced in such away that at any one moment each is transcribing a differentportion of the DNA sequence. An electron micrographof extremely active RNA synthesis is shownin Figure 37–4.Figure 37–4. Electron photomicrograph of multiplecopies of amphibian ribosomal RNA genes in theprocess of being transcribed. The magnification isabout 6000 ×. Note that the length of the transcripts increasesas the RNA polymerase molecules progressalong the individual ribosomal RNA genes; transcriptionstart sites (filled circles) to transcription terminationsites (open circles). RNA polymerase I (not visualizedhere) is at the base of the nascent rRNA transcripts.Thus, the proximal end of the transcribed gene hasshort transcripts attached to it, while much longer transcriptsare attached to the distal end of the gene. Thearrows indicate the direction (5′ to 3′) of transcription.(Reproduced with permission, from Miller OL Jr, Beatty BR:Portrait of a gene. J Cell Physiol 1969;74[Suppl 1]:225.)THE FIDELITY & FREQUENCY OFTRANSCRIPTION IS CONTROLLEDBY PROTEINS BOUND TO CERTAINDNA SEQUENCESThe DNA sequence analysis of specific genes has allowedthe recognition of a number of sequences importantin gene transcription. From the large number ofbacterial genes studied it is possible to construct consensusmodels of transcription initiation and terminationsignals.