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RNA Polymerase II Elongation Control in Eukaryotes
RNA Polymerase II Elongation Control in Eukaryotes D H Price, University of Iowa, Iowa City, IA, USA Published by Elsevier Inc. This article is reproduced from the previous edition, volume 3, pp. 766–769, ã 2004, Elsevier Inc.
Glossary DRB The P-TEFb inhibitor 5,6-dichlororibofuranosyl benzamidizole. DSIF DRB sensitivity inducing factor, an N-TEF.
During the 1990s, it became clear that the elongation properties of RNA polymerase II are regulated and the factors responsible play a critical role in controlling gene expression in eukaryotic cells. One set of factors helps RNA polymerase II to negotiate pause and arrest sequences in the template, and to maintain an elongation rate of 1000–1500 nucleotides per minute. The initiation factor TFIIF (transcription initiation factor IIF) reduces RNA polymerase II pause times and stimulates the rate of elongation. The elongation factor S-II or transcription initiation factor IIS (TFIIS) rescues polymerase molecules that are blocked from further elongation at arrest sites. Another set of factors is responsible for an elongation control process that regulates the fraction of initiated polymerases that enter productive elongation to generate messenger RNAs (mRNAs). Negative transcription elongation factors (N-TEFs), such as 5,6-dichlororibofuranosyl benzamidizole (DRB) sensitivity inducing factor (DSIF) and negative elongation factor (NELF), slow the rate of elongation and restrict the polymerase to promoter proximal sequences. The positive transcription elongation factor, P-TEFb, overcomes the effect of the negative factors and allows the production of full-length transcripts. The function of P-TEFb is regulated through interactions with many other transcription factors and the amount of active P-TEFb in the cell is precisely regulated through a reversible inhibition by the small cellular RNA, 7SK.
Historical Perspective It has been clear for some time that much of the regulation of prokaryotic gene expression is accomplished by controlling the elongation potential of RNA polymerase, but only recently has it become apparent that eukaryotic gene expression is similarly regulated. Early work in John Lis’s lab showed that there was an RNA polymerase II molecule engaged in transcription of the major Drosophila heat shock gene, HSP70, under conditions where no full-length mRNAs were being produced. Only after heat shock were the polymerases allowed to continue elongation and produce mRNAs. Similarly, regulated premature termination of transcription was seen on a number of viral genes, most notably from human immuno-deficiency virus (HIV), as well as on cellular genes, including oncogenes such as c-myc and c-fos. In the early 1990s, in vitro systems were beginning to reproduce the elongation control process and ultimately these
164
NELF Negative elongation factor, an N-TEF. N-TEFs Negative transcription elongation factors. P-TEFb Positive transcription elongation factor b composed of Cdk9 and a cyclin partner.
systems allowed the identification and purification of some of the specific factors involved. The current model of RNA polymerase II elongation control has the look and feel of models of prokaryotic antitermination, but there are important differences in mechanistic details.
Positive and Negative Elongation Factors Elongation Maintenance Factors During transcription, RNA polymerase II is aided by specific factors as it encounters numerous blocks to elongation inherent in a template sequence. During normal transcription in vivo, each nucleotide addition to an RNA chain requires 50 ms on average. However, in vitro RNA polymerase II may pause for seconds or minutes at specific sites in the absence of any accessory factors. TFIIF, a factor required for RNA polymerase II initiation, can also decrease the time that the polymerase spends at pause sites. This has the effect of increasing the overall elongation rate because, for the most part, the elongation rate is determined by how long the polymerase stops at the strongest pause sites. It has been hypothesized that the mechanism utilized by TFIIF involves an interaction-induced change in the polymerase from the paused conformation to the elongation competent form. Other factors that belong in this same class are eleven-nineteen lysine-rich leukemia and elongin, but TFIIF has the most dramatic elongation stimulatory activity. The elongation factor S-II functions by a completely different mechanism. At some sites a fraction of RNA polymerase II molecules fall into an arrested conformation from which they cannot escape unaided. At these sites the 30 end of the nascent transcript is removed from the active site of the polymerase as the polymerase backslides along the template while maintaining an RNA:DNA hybrid. When this happens, the polymerase may remain engaged for hours or days without extending the transcript. S-II stimulates an intrinsic ribonuclease activity of the polymerase that removes the unpaired 30 end of the transcript and puts the new 30 end in register with the active site of the polymerase. This reactivates elongation and the polymerase has a second chance to pass the arrest site. Eventually, all polymerases can pass the arrest site, although many may require several rounds of S-II-mediated transcript cleavage. Through the combined function of both classes of factors, the elongation rate of RNA polymerase II is maintained between 1000 and 1500 nucleotides per minute.
Molecular Biology | RNA Polymerase II Elongation Control in Eukaryotes
P P P P P P
Productive elongation P-TEFb CTD
DSIF
PoI II
RNA
TTF2
NELF
Abortive elongation
Figure 1 RNA polymerase II elongation control. After initiation, pol II enters abortive elongation by default because of the action of N-TEFs, such as DSIF and NELF. Phosphorylation of the CTD of the large subunit of RNA polymerase II by P-TEFb causes the transition into productive elongation. If P-TEFb does not act, transcription is prematurely terminated by TTF2.
Elongation Control In addition to the factors that maintain efficient elongation, there is another set of factors responsible for regulating the efficiency of initiated polymerases to reach the 30 end of genes. This process has been termed ‘RNA polymerase II elongation control’ and is best viewed as an obligatory event mediated by the default action of negative factors, N-TEFs, and the regulated action of a positive factor, P-TEFb (Figure 1). The process was demonstrated in vitro by first showing that the transcription inhibitor DRB could block the production of long, but not short, transcripts. Elongation control was further characterized by the separation of N-TEFs, which direct polymerases to generate only short promoter proximal transcripts, from a positive factor, ultimately identified as P-TEFb, which reverses the action of the N-TEFs. A combination of the action of the N-TEFs and the activity of transcription termination factor 2 (TTF2) results in a process called ‘abortive elongation’ characterized by the premature termination of transcripts before the mature 30 end of the gene is reached. P-TEFb plays a key role in that it is responsible for the transition into productive elongation. Stated in another way, the ability to produce mRNAs is directly linked to the activity of P-TEFb. Elongation control machinery exists in all eukaryotic species that have been examined and the current evidence suggests that P-TEFb is required for transcription of most genes.
N-TEFs and Termination Factors The evidence gathered so far points to the existence of two factors (DSIF and NELF) that play a negative role in elongation control. DSIF, the DRBIF, was discovered as a protein required for DRB sensitive transcription in vitro. It is comprised of two subunits with strong sequence similarity to the yeast proteins SPT4 and SPT5. DSIF has almost no function on RNA polymerase II alone. The NELF also has no effect on RNA polymerase II alone, but the combination of NELF and DSIF is able to slow the elongation rate and cause the polymerase to reside longer at pause sites. In this way, NELF and DSIF act in exactly the opposite way as TFIIF, which reduces pause time. NELF is comprised of four subunits, including a
165
putative RNA-binding protein called RD. A defined elongation control system comprised of isolated RNA polymerase II elongation complexes (NELF, DSIF, and P-TEFb) displays many of the properties of elongation control seen when transcription is carried out in nuclear extract. DSIF is involved in controlling transcription from the HSP70 locus in Drosophila, but the generality of its function has not been demonstrated. Although removal of DSIF in Caenorhabditis elegans using RNA interference techniques relieved most of the requirements of P-TEFb at a heat shock locus, similar results were not found in expression of several other genes. These facts along with the fact that homologs of NELF subunits have not been identified in many organisms suggest that other negative factors, which operate at a wide variety of genes, remain to be discovered. So far only one transcription termination factor, TTF2 (formerly and uninformatively called factor 2), has been identified. It is a member of the SWI2/SNF2 family of proteins that are generally involved in disrupting protein nucleic acid interactions. It binds to both single- and double-strand (ds) DNA and has a strong dsDNA-dependent adenosine triphosphatase activity that is required for termination activity. The termination activity of TTF2 is inhibited by TFIIF, and while the mechanism of this inhibition is unknown, it provides a possible method to control the activity of TTF2 during transcription. The role of TTF2 seems to operate outside of the elongation control process in that its negative effect is not directly reversed by P-TEFb. However, a role for the factor in abortive elongation (premature termination) or in normal termination associated with mature 30 end formation has not been ruled out.
P-TEFb Subunits and Activity P-TEFb is a cyclin-dependent kinase comprised of Cdk9 and one of several cyclin subunits. Three genes in humans – T1, T2, and K – encode cyclin subunits that can associate with and activate Cdk9. Two cyclin T genes have been found in C. elegans, but only one in Drosophila. Supporting a broad role in gene expression, knockout of Cdk9 in C. elegans causes loss of gene expression and death at the same early stage as knockout of one of the subunits of RNA polymerase II. The same lethal phenotype is observed when both cyclin Ts are knocked out. The carboxyl terminal domain (CTD) of the large subunit of RNA polymerase II is phosphorylated by P-TEFb, and this domain has been shown to be required for the function of P-TEFb in the transition into productive elongation. The CTD is comprised of multiple repeats of the heptapeptide YSPTSPS, and P-TEFb predominately phosphorylates the second serine. P-TEFb had been shown to have broad substrate specificity in vitro and will phosphorylate the large subunit of both DSIF and TFIIF. Although the CTD is phosphorylated by P-TEFb and is required for the function of P-TEFb in transcription, there may be other important functional phosphorylation targets. The effect of DRB on transcription is explained by the fact that it inhibits the kinase activity of P-TEFb. All P-TEFb inhibitors reduce the production of mRNAs in vivo and are lethal at high concentrations. Flavopiridol, the most potent P-TEFb inhibitor found, is in clinical trials against cancer.
166
Molecular Biology | RNA Polymerase II Elongation Control in Eukaryotes
Recruitment of P-TEFb P-TEFb interacts in a functional way with a number of important transcription factors. Cyclin T1 interacts with the HIV transactivator Tat, and P-TEFb containing cyclin T1 can be recruited to elongation complexes on the HIV template through the interaction of Tat and cyclin T1 with a stem–loop structure that forms in the nascent HIV transcript called ‘transactivation response (TAR)’. This recruitment of P-TEFb activates transcription of the HIV genome. A cellular factor, CIITA, which is involved in activating transcription of the major histocompatibility complex (MHC) class II genes, has also been shown to function through interaction with P-TEFb. That interaction is blocked in the presence of Tat suggesting that the binding site is similar for the viral and cellular protein and providing a mechanism for inhibition of MHC class II gene expression during HIV infection. Other transcription factors and enhancer binding proteins – including nuclear factor kappa B, c-myc, major Cdk9-interacting elongation factor, and the androgen receptor – have been demonstrated to various degrees to have functional interactions with P-TEFb. Recruitment of P-TEFb through artificial targeting to promoter DNA or to nascent RNA has revealed several important aspects of its function. First, when a protein is expressed in which Cdk9 or cyclin T1 or T2 is tethered to the DNA-binding domain of a yeast protein (Gal4) and the binding sites for that protein are placed close to a human promoter, transcription is stimulated from that promoter. Besides the core promoter elements, SP1 binding sites are also required to see an effect of the recruited P-TEFb in this system. This is consistent with the model for P-TEFb function because P-TEFb only affects polymerases that have already initiated, and SP1 increases the initiation efficiency. Expression of Cdk9, cyclin T1, T2, or K tethered to an RNA-binding domain (e.g., HIV rev) activates transcription of a gene containing the appropriate RNA element. In both tethering experiments, the untethered component of P-TEFb is also recruited due to the inherent interaction between P-TEFb subunits. Cyclin K does not work when tethered to DNA elements because it lacks a CTD present on both cyclin T1 and T2 that plays another role by interacting with the CTD of the large subunit of RNA polymerase II.
Control of P-TEFb by 7SK Very recently, the laboratories of Olivier Bensaude and Qiang Zhou simultaneously discovered the small cellular RNA, 7SK, in association with a fraction of P-TEFb in cells. The large complex containing P-TEFb and 7SK was relatively inactive compared to the free P-TEFb (Figure 2). Addition of small amounts of compounds that inhibit elongation by RNA polymerase II causes a reduction in the large form of P-TEFb in vivo and a concomitant increase in the small active form. Evidently, a cell will try to compensate for reduced production of mRNAs by activating more P-TEFb. The change in large to small form of P-TEFb can occur within minutes and is reversible. The significance of the large form of P-TEFb was demonstrated using a mouse model of cardiac hypertrophy. Heart cells that were stimulated to grow in size had an increase in P-TEFb activity, but no increase in the amount of P-TEFb. It was shown that all hypertrophic signals tested caused a release of 7SK. The signal transduction pathways involved in hypertrophy of cardiac cells at least partially routes through the elongation control pathway.
+
Active
7SK
Inactive
Figure 2 Control of P-TEFb by 7SK. The amount of P-TEFb activity in the cell can be quickly adjusted by the reversible association of 7SK RNA. The large form with 7SK bound is inactive.
Integration of Elongation Control and Gene Expression Elongation control may play a very significant role in controlling eukaryotic gene expression. Most RNA polymerase II molecules found transcribing human genes require the prior function of P-TEFb. Recruitment of P-TEFb by transcription factors allows specific genes to be targeted, and both the targeted and the untargeted functions of P-TEFb may allow global regulation of mRNA production. Regulating the conversion of the large to small P-TEFb form by signal transduction pathways is another global means to regulate mRNA levels. Overall, the combined action of negative and then positive factors results in a kinetic delay of the progression of polymerases down the gene. This may provide a window of opportunity for RNAprocessing machinery to function or associate with the elongating polymerase, ensuring a truly productive elongation event resulting in a complete, functional mRNA. The human capping enzyme is functionally coupled to transcription and guanylates the nascent transcript before P-TEFb acts. However, some components of the polyadenylation and splicing machinery have been shown to associate with the phosphorylated CTD. Perhaps the processing machinery is functionally coupled to transcription through interactions provided by elongation control and scans the nascent transcript for processing sites as it is being made. Many mechanistic details of how RNA polymerase II elongation control is accomplished and the ramifications of the process on subsequent events remain to be determined.
See also: Molecular Biology: RNA Polymerase II and Its General Transcription Factors; Signaling: Tachykinin/Substance P/ Neurokinin-1 Receptors.
Further Reading Marshall NF and Price DH (1992) Control of formation of two distinct classes of RNA polymerase II elongation complexes. Molecular and Cellular Biology 12: 2078–2090. Nguyen VT, Kiss T, Michels AA, and Bensaude O (2001) 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414: 322–325. Price DH (2000) P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Molecular and Cellular Biology 20: 2629–2634. Sano M, Abdellatif M, Oh H, et al. (2002) Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. Nature Medicine 8: 1310–1317. Shim EY, Walker AK, Shi Y, and Blackwell TK (2002) CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. Genes and Development 16: 2135–2146. Zhu Y, Pe’ery T, Peng J, et al. (1997) Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes and Development 11: 2622–2632.
Glossary DRB The P-TEFb inhibitor 5,6-dichlororibofuranosyl benzamidizole. DSIF DRB sensitivity inducing factor, an N-TEF.
During the 1990s, it became clear that the elongation properties of RNA polymerase II are regulated and the factors responsible play a critical role in controlling gene expression in eukaryotic cells. One set of factors helps RNA polymerase II to negotiate pause and arrest sequences in the template, and to maintain an elongation rate of 1000–1500 nucleotides per minute. The initiation factor TFIIF (transcription initiation factor IIF) reduces RNA polymerase II pause times and stimulates the rate of elongation. The elongation factor S-II or transcription initiation factor IIS (TFIIS) rescues polymerase molecules that are blocked from further elongation at arrest sites. Another set of factors is responsible for an elongation control process that regulates the fraction of initiated polymerases that enter productive elongation to generate messenger RNAs (mRNAs). Negative transcription elongation factors (N-TEFs), such as 5,6-dichlororibofuranosyl benzamidizole (DRB) sensitivity inducing factor (DSIF) and negative elongation factor (NELF), slow the rate of elongation and restrict the polymerase to promoter proximal sequences. The positive transcription elongation factor, P-TEFb, overcomes the effect of the negative factors and allows the production of full-length transcripts. The function of P-TEFb is regulated through interactions with many other transcription factors and the amount of active P-TEFb in the cell is precisely regulated through a reversible inhibition by the small cellular RNA, 7SK.
Historical Perspective It has been clear for some time that much of the regulation of prokaryotic gene expression is accomplished by controlling the elongation potential of RNA polymerase, but only recently has it become apparent that eukaryotic gene expression is similarly regulated. Early work in John Lis’s lab showed that there was an RNA polymerase II molecule engaged in transcription of the major Drosophila heat shock gene, HSP70, under conditions where no full-length mRNAs were being produced. Only after heat shock were the polymerases allowed to continue elongation and produce mRNAs. Similarly, regulated premature termination of transcription was seen on a number of viral genes, most notably from human immuno-deficiency virus (HIV), as well as on cellular genes, including oncogenes such as c-myc and c-fos. In the early 1990s, in vitro systems were beginning to reproduce the elongation control process and ultimately these
164
NELF Negative elongation factor, an N-TEF. N-TEFs Negative transcription elongation factors. P-TEFb Positive transcription elongation factor b composed of Cdk9 and a cyclin partner.
systems allowed the identification and purification of some of the specific factors involved. The current model of RNA polymerase II elongation control has the look and feel of models of prokaryotic antitermination, but there are important differences in mechanistic details.
Positive and Negative Elongation Factors Elongation Maintenance Factors During transcription, RNA polymerase II is aided by specific factors as it encounters numerous blocks to elongation inherent in a template sequence. During normal transcription in vivo, each nucleotide addition to an RNA chain requires 50 ms on average. However, in vitro RNA polymerase II may pause for seconds or minutes at specific sites in the absence of any accessory factors. TFIIF, a factor required for RNA polymerase II initiation, can also decrease the time that the polymerase spends at pause sites. This has the effect of increasing the overall elongation rate because, for the most part, the elongation rate is determined by how long the polymerase stops at the strongest pause sites. It has been hypothesized that the mechanism utilized by TFIIF involves an interaction-induced change in the polymerase from the paused conformation to the elongation competent form. Other factors that belong in this same class are eleven-nineteen lysine-rich leukemia and elongin, but TFIIF has the most dramatic elongation stimulatory activity. The elongation factor S-II functions by a completely different mechanism. At some sites a fraction of RNA polymerase II molecules fall into an arrested conformation from which they cannot escape unaided. At these sites the 30 end of the nascent transcript is removed from the active site of the polymerase as the polymerase backslides along the template while maintaining an RNA:DNA hybrid. When this happens, the polymerase may remain engaged for hours or days without extending the transcript. S-II stimulates an intrinsic ribonuclease activity of the polymerase that removes the unpaired 30 end of the transcript and puts the new 30 end in register with the active site of the polymerase. This reactivates elongation and the polymerase has a second chance to pass the arrest site. Eventually, all polymerases can pass the arrest site, although many may require several rounds of S-II-mediated transcript cleavage. Through the combined function of both classes of factors, the elongation rate of RNA polymerase II is maintained between 1000 and 1500 nucleotides per minute.
Molecular Biology | RNA Polymerase II Elongation Control in Eukaryotes
P P P P P P
Productive elongation P-TEFb CTD
DSIF
PoI II
RNA
TTF2
NELF
Abortive elongation
Figure 1 RNA polymerase II elongation control. After initiation, pol II enters abortive elongation by default because of the action of N-TEFs, such as DSIF and NELF. Phosphorylation of the CTD of the large subunit of RNA polymerase II by P-TEFb causes the transition into productive elongation. If P-TEFb does not act, transcription is prematurely terminated by TTF2.
Elongation Control In addition to the factors that maintain efficient elongation, there is another set of factors responsible for regulating the efficiency of initiated polymerases to reach the 30 end of genes. This process has been termed ‘RNA polymerase II elongation control’ and is best viewed as an obligatory event mediated by the default action of negative factors, N-TEFs, and the regulated action of a positive factor, P-TEFb (Figure 1). The process was demonstrated in vitro by first showing that the transcription inhibitor DRB could block the production of long, but not short, transcripts. Elongation control was further characterized by the separation of N-TEFs, which direct polymerases to generate only short promoter proximal transcripts, from a positive factor, ultimately identified as P-TEFb, which reverses the action of the N-TEFs. A combination of the action of the N-TEFs and the activity of transcription termination factor 2 (TTF2) results in a process called ‘abortive elongation’ characterized by the premature termination of transcripts before the mature 30 end of the gene is reached. P-TEFb plays a key role in that it is responsible for the transition into productive elongation. Stated in another way, the ability to produce mRNAs is directly linked to the activity of P-TEFb. Elongation control machinery exists in all eukaryotic species that have been examined and the current evidence suggests that P-TEFb is required for transcription of most genes.
N-TEFs and Termination Factors The evidence gathered so far points to the existence of two factors (DSIF and NELF) that play a negative role in elongation control. DSIF, the DRBIF, was discovered as a protein required for DRB sensitive transcription in vitro. It is comprised of two subunits with strong sequence similarity to the yeast proteins SPT4 and SPT5. DSIF has almost no function on RNA polymerase II alone. The NELF also has no effect on RNA polymerase II alone, but the combination of NELF and DSIF is able to slow the elongation rate and cause the polymerase to reside longer at pause sites. In this way, NELF and DSIF act in exactly the opposite way as TFIIF, which reduces pause time. NELF is comprised of four subunits, including a
165
putative RNA-binding protein called RD. A defined elongation control system comprised of isolated RNA polymerase II elongation complexes (NELF, DSIF, and P-TEFb) displays many of the properties of elongation control seen when transcription is carried out in nuclear extract. DSIF is involved in controlling transcription from the HSP70 locus in Drosophila, but the generality of its function has not been demonstrated. Although removal of DSIF in Caenorhabditis elegans using RNA interference techniques relieved most of the requirements of P-TEFb at a heat shock locus, similar results were not found in expression of several other genes. These facts along with the fact that homologs of NELF subunits have not been identified in many organisms suggest that other negative factors, which operate at a wide variety of genes, remain to be discovered. So far only one transcription termination factor, TTF2 (formerly and uninformatively called factor 2), has been identified. It is a member of the SWI2/SNF2 family of proteins that are generally involved in disrupting protein nucleic acid interactions. It binds to both single- and double-strand (ds) DNA and has a strong dsDNA-dependent adenosine triphosphatase activity that is required for termination activity. The termination activity of TTF2 is inhibited by TFIIF, and while the mechanism of this inhibition is unknown, it provides a possible method to control the activity of TTF2 during transcription. The role of TTF2 seems to operate outside of the elongation control process in that its negative effect is not directly reversed by P-TEFb. However, a role for the factor in abortive elongation (premature termination) or in normal termination associated with mature 30 end formation has not been ruled out.
P-TEFb Subunits and Activity P-TEFb is a cyclin-dependent kinase comprised of Cdk9 and one of several cyclin subunits. Three genes in humans – T1, T2, and K – encode cyclin subunits that can associate with and activate Cdk9. Two cyclin T genes have been found in C. elegans, but only one in Drosophila. Supporting a broad role in gene expression, knockout of Cdk9 in C. elegans causes loss of gene expression and death at the same early stage as knockout of one of the subunits of RNA polymerase II. The same lethal phenotype is observed when both cyclin Ts are knocked out. The carboxyl terminal domain (CTD) of the large subunit of RNA polymerase II is phosphorylated by P-TEFb, and this domain has been shown to be required for the function of P-TEFb in the transition into productive elongation. The CTD is comprised of multiple repeats of the heptapeptide YSPTSPS, and P-TEFb predominately phosphorylates the second serine. P-TEFb had been shown to have broad substrate specificity in vitro and will phosphorylate the large subunit of both DSIF and TFIIF. Although the CTD is phosphorylated by P-TEFb and is required for the function of P-TEFb in transcription, there may be other important functional phosphorylation targets. The effect of DRB on transcription is explained by the fact that it inhibits the kinase activity of P-TEFb. All P-TEFb inhibitors reduce the production of mRNAs in vivo and are lethal at high concentrations. Flavopiridol, the most potent P-TEFb inhibitor found, is in clinical trials against cancer.
166
Molecular Biology | RNA Polymerase II Elongation Control in Eukaryotes
Recruitment of P-TEFb P-TEFb interacts in a functional way with a number of important transcription factors. Cyclin T1 interacts with the HIV transactivator Tat, and P-TEFb containing cyclin T1 can be recruited to elongation complexes on the HIV template through the interaction of Tat and cyclin T1 with a stem–loop structure that forms in the nascent HIV transcript called ‘transactivation response (TAR)’. This recruitment of P-TEFb activates transcription of the HIV genome. A cellular factor, CIITA, which is involved in activating transcription of the major histocompatibility complex (MHC) class II genes, has also been shown to function through interaction with P-TEFb. That interaction is blocked in the presence of Tat suggesting that the binding site is similar for the viral and cellular protein and providing a mechanism for inhibition of MHC class II gene expression during HIV infection. Other transcription factors and enhancer binding proteins – including nuclear factor kappa B, c-myc, major Cdk9-interacting elongation factor, and the androgen receptor – have been demonstrated to various degrees to have functional interactions with P-TEFb. Recruitment of P-TEFb through artificial targeting to promoter DNA or to nascent RNA has revealed several important aspects of its function. First, when a protein is expressed in which Cdk9 or cyclin T1 or T2 is tethered to the DNA-binding domain of a yeast protein (Gal4) and the binding sites for that protein are placed close to a human promoter, transcription is stimulated from that promoter. Besides the core promoter elements, SP1 binding sites are also required to see an effect of the recruited P-TEFb in this system. This is consistent with the model for P-TEFb function because P-TEFb only affects polymerases that have already initiated, and SP1 increases the initiation efficiency. Expression of Cdk9, cyclin T1, T2, or K tethered to an RNA-binding domain (e.g., HIV rev) activates transcription of a gene containing the appropriate RNA element. In both tethering experiments, the untethered component of P-TEFb is also recruited due to the inherent interaction between P-TEFb subunits. Cyclin K does not work when tethered to DNA elements because it lacks a CTD present on both cyclin T1 and T2 that plays another role by interacting with the CTD of the large subunit of RNA polymerase II.
Control of P-TEFb by 7SK Very recently, the laboratories of Olivier Bensaude and Qiang Zhou simultaneously discovered the small cellular RNA, 7SK, in association with a fraction of P-TEFb in cells. The large complex containing P-TEFb and 7SK was relatively inactive compared to the free P-TEFb (Figure 2). Addition of small amounts of compounds that inhibit elongation by RNA polymerase II causes a reduction in the large form of P-TEFb in vivo and a concomitant increase in the small active form. Evidently, a cell will try to compensate for reduced production of mRNAs by activating more P-TEFb. The change in large to small form of P-TEFb can occur within minutes and is reversible. The significance of the large form of P-TEFb was demonstrated using a mouse model of cardiac hypertrophy. Heart cells that were stimulated to grow in size had an increase in P-TEFb activity, but no increase in the amount of P-TEFb. It was shown that all hypertrophic signals tested caused a release of 7SK. The signal transduction pathways involved in hypertrophy of cardiac cells at least partially routes through the elongation control pathway.
+
Active
7SK
Inactive
Figure 2 Control of P-TEFb by 7SK. The amount of P-TEFb activity in the cell can be quickly adjusted by the reversible association of 7SK RNA. The large form with 7SK bound is inactive.
Integration of Elongation Control and Gene Expression Elongation control may play a very significant role in controlling eukaryotic gene expression. Most RNA polymerase II molecules found transcribing human genes require the prior function of P-TEFb. Recruitment of P-TEFb by transcription factors allows specific genes to be targeted, and both the targeted and the untargeted functions of P-TEFb may allow global regulation of mRNA production. Regulating the conversion of the large to small P-TEFb form by signal transduction pathways is another global means to regulate mRNA levels. Overall, the combined action of negative and then positive factors results in a kinetic delay of the progression of polymerases down the gene. This may provide a window of opportunity for RNAprocessing machinery to function or associate with the elongating polymerase, ensuring a truly productive elongation event resulting in a complete, functional mRNA. The human capping enzyme is functionally coupled to transcription and guanylates the nascent transcript before P-TEFb acts. However, some components of the polyadenylation and splicing machinery have been shown to associate with the phosphorylated CTD. Perhaps the processing machinery is functionally coupled to transcription through interactions provided by elongation control and scans the nascent transcript for processing sites as it is being made. Many mechanistic details of how RNA polymerase II elongation control is accomplished and the ramifications of the process on subsequent events remain to be determined.
See also: Molecular Biology: RNA Polymerase II and Its General Transcription Factors; Signaling: Tachykinin/Substance P/ Neurokinin-1 Receptors.
Further Reading Marshall NF and Price DH (1992) Control of formation of two distinct classes of RNA polymerase II elongation complexes. Molecular and Cellular Biology 12: 2078–2090. Nguyen VT, Kiss T, Michels AA, and Bensaude O (2001) 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414: 322–325. Price DH (2000) P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Molecular and Cellular Biology 20: 2629–2634. Sano M, Abdellatif M, Oh H, et al. (2002) Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. Nature Medicine 8: 1310–1317. Shim EY, Walker AK, Shi Y, and Blackwell TK (2002) CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. Genes and Development 16: 2135–2146. Zhu Y, Pe’ery T, Peng J, et al. (1997) Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes and Development 11: 2622–2632.