Eukaryotic termination and polyadenylation
Unlike prokaryotes that have a single RNA polymerase for transcription, eukaryotes have three RNA polymerases (Polymerases I, II, and III), each responsible for transcribing different types of RNA: Polymerase I is responsible for ribosomal RNA, Polymerase II is responsible for mRNA and miRNAs, and Polymerase III transcribes tRNA and other short RNAs. Although not as well studied as prokaryotic termination, the basic processes for eukaryotic termination are understood and it has been noted that each eukaryotic RNA polymerase terminates differently. Polymerase III, for example, relies on a specific sequence and RNA secondary structure to induce transcript cleavage, similar to the Rho-independent termination found in prokaryotes. This is different than Polymerases I and II, which both require binding of termination factors. Although both are termination factor dependent, Polymerases I and II employ different mechanisms to terminate transcription. Polymerase I uses a process similar to the prokaryotic Rho-dependent mechanism, whereas Polymerase II termination is more complex and involves two RNA polymerase-associated proteins, CPSF and CstF, which are responsible for recruiting the cleavage and polyadenylation enzymes, in a process that seems to couple termination with polyadenlyation.
Mammalian expression plasmids are primarily used to create mRNA and the commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) include the sequence motif AAUAAA which promotes both polyadenylation and termination. Out of those listed, the SV40 late polyA and rbGlob polyA are thought to be more efficient in terminating transcription due to the presence of additional helper sequences (2-3).
As alluded to above, termination and polyadenylation of Polymerase II transcripts (and therefore mRNAs) are coordinated processes. Cleavage between the consensus motif and a downstream GU-rich region (shown in the figure below) releases the mRNA from the polymerase and creates a free 3' end which is now available for polyadenylation. The addition of the poly(A) tail is important for stability of the mRNA, protection from degradation, and is integral to the nuclear export and translation processes as well.
Figure 2: The conserved eukaryotic polyadenylation signal directs cleavage at the cleave signal and addition of a poly-A tail to the mRNA transcript. By Arunreginald at en.wikipedia, CC BY-SA
