Regulated Elongation and Termination of Transcription

In eukaryotes, the mechanisms for terminating transcription differ for each of the three RNA polymerases. Transcription of pre-rRNA genes by RNA polymerase I is terminated by a mechanism that requires a polymerase-specific termination factor. This DNA-binding protein binds to a specific DNA sequence downstream of the transcription unit. Efficient termination requires that the termination factor bind to the template DNA in the correct orientation. Purified RNA polymerase III terminates after polymerizing a series of U residues. The deoxy(A)n-ribo(U)n DNA-RNA hybrid that results when a stretch of U's are synthesized is particularly unstable compared with all other base-paired sequences. The ease with which this hybrid can be melted probably contributes to the mechanism of termination by RNA polymerase III.

In most mammalian protein-coding genes transcribed by RNA polymerase II, once the polymerase has transcribed beyond about fifty bases, further elongation is highly processive and does not terminate until after a sequence is transcribed that directs cleavage and polyadenylation of the RNA at the sequence that forms the 3' end of the encoded mRNA. RNA polymerase II then can terminate at multiple sites located over a distance of 0.5-2 kb beyond this poly(A) addition site. Experiments with mutant genes show that termination is coupled to the process that cleaves and polyadenylates the 3' end of a transcript, which is discussed in the next chapter. Biochemical and chromatin immunopre-cipitation experiments suggest that the protein complex that cleaves and polyadenylates the nascent mRNA transcript at specific sequences associates with the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II following initiation (see Figure 11-27). This cleavage/ polyadenylation complex may suppress termination by RNA polymerase II until the sequence signaling cleavage and polyadenylation is transcribed by the polymerase.

While transcription termination is unregulated for most genes, for some specific genes, a choice is made between elongation and termination or pausing within a few tens of bases from the transcription start site. This choice between elongation and termination or pausing can be regulated; thus expression of the encoded protein is controlled not only by transcription initiation, but also by control of transcription elongation early in the transcription unit. We discuss two examples of such regulation next.

Transcription of the HIV Genome Is Regulated by an Antitermination Mechanism

Currently, transcription of the human immunodeficiency virus (HIV) genome by RNA polymerase II provides the best-understood example of regulated transcription termination in eukaryotes. Efficient expression of HIV genes requires a small viral protein encoded at the tat locus. Cells infected with tat~ mutants produce short viral transcripts that hybridize to restriction fragments containing promoter-proximal regions of the HIV DNA but not to restriction fragments farther downstream from the promoter. In contrast, cells infected with wild-type HIV synthesize long viral transcripts that hybridize to restriction fragments throughout the single HIV transcription unit. Thus Tat protein functions as an antitermination factor, permitting RNA polymerase II to read through a transcriptional block. Since antitermination by Tat protein is required for HIV replication, further understanding of this gene-control mechanism may offer possibilities for designing effective therapies for acquired immunodeficiency syndrome (AIDS).

Tat is a sequence-specific RNA-binding protein. It binds to the RNA copy of a sequence called TAR, which is located near the 5' end of the HIV transcript. The TAR sequence folds into an RNA hairpin with a bulge in the middle of the stem (Figure 11-45). TAR contains two binding sites: one that interacts with Tat and one that interacts with a cellular protein called cyclin T. As depicted in Figure 11-45, the HIV Tat protein and cellular cyclin T each bind to TAR RNA and also interact directly with each other so that they bind cooperatively, much like the cooperative binding of DNA-binding transcription factors (see Figure 11-24). Interaction of cyclin T with a protein kinase called CDK9 activates the kinase, whose substrate is the CTD of RNA polymerase II. In vitro transcription studies using a specific inhibitor of CDK9 suggest that RNA poly-merase II molecules that initiate transcription on the HIV promoter terminate after transcribing «50 bases unless the CTD is hyperphosphorylated by CDK9. Cooperative binding of cyclin T and Tat to the TAR sequence at the 5' end of the HIV transcript positions CDK9 so that it can phosphorylate the CTD, thereby preventing termination and permitting the polymerase to continue chain elongation.

▲ FIGURE 11-45 Model of antitermination complex composed of HIV Tat protein and several cellular proteins.

The TAR element In the HIV transcript contains sequences recognized by Tat and the cellular protein cyclin T Cyclin T activates and helps position the protein kinase CDK9 near its substrate, the CTD of RNA polymerase II. See the text for a more detailed discussion. [See P Wei et al., 1998, Cell 92:451; T Wada et al., 1998, Genes & Devel. 12:357; and Y Yamaguchi et al., 1999, Cell 97:41.]

Several additional cellular proteins, including Spt4 and Spt5 and the NELF complex, participate in the process by which HIV Tat controls elongation versus termination (see Figure 11-45). Experiments with the specific inhibitor of CDK9 mentioned above and with spt4 and spt5 yeast mutants indicate that these cellular proteins are required for transcription elongation beyond «50 bases for most cellular genes. But for most genes, these proteins appear to function constitutively, that is, without being regulated. As discussed in Chapter 12, RNA polymerase II pausing instigated by Spt4/5 and NELF is thought to delay elongation until mRNA processing factors associate with the phosphorylated CTD. Further phosphorylation of the CTD by cyclin T-CDK9 (also known as pTEFb) appears to reverse this pause and allow elongation to continue. Currently, it is not clear why this process is not constitutive for the HIV promoter, where cooperative binding of HIV Tat and cyclin T to the TAR RNA sequence is required for efficient elongation.

Promoter-Proximal Pausing of RNA Polymerase II Occurs in Some Rapidly Induced Genes

The heat-shock genes (e.g., hsp70) illustrate another mechanism for regulating RNA chain elongation in eukaryotes. During transcription of these genes, RNA polymerase II pauses after transcribing «25 nucleotides but does not terminate transcription (as it does when transcribing the HIV genome in the absence of Tat protein). The paused poly-merase remains associated with the nascent RNA and template DNA, until conditions occur that lead to activation of HSTF (heat-shock transcription factor). Subsequent binding of activated HSTF to specific sites in the promoter-proximal region of heat-shock genes stimulates the paused polymer-ase to continue chain elongation and promotes rapid reinitiation by additional RNA polymerase II molecules.

The pausing during transcription of heat-shock genes initially was discovered in Drosophila, but a similar mechanism most likely occurs in other eukaryotes. Heat-shock genes are induced by intracellular conditions that denature proteins (such as elevated temperature, "heat shock"). Some encode proteins that are relatively resistant to denaturing conditions and act to protect other proteins from denaturation; others are chaperonins that refold denatured proteins (see Chapter 3). The mechanism of transcriptional control that evolved to regulate expression of these genes permits a rapid response: these genes are already paused in a state of suspended transcription and therefore, when an emergency arises, require no time to remodel and acetylate chromatin over the promoter and assemble a transcription preinitiation complex.

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