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human mediator figure 12-17 Comparison of the Yeast and Human Mediators. The homologous proteins are shown in dart: btue (Source: Modified with permission from Malik S and Roeder R. C. 2000 Transcriptional regulation through mediator-like coectivators in yeast and metazoan cetfs. Trends Bivctiem. Set 25: 277-2BS Copyright e 2000, With permission from Elsevier.)

different groups of regulators (activators and repressors). It is equally possible, however, that the variations seen in subunil composition are artifacts, simply reflecting different methods of isolation.

In some studies it has been shown that a complex consisting of Pol II, Mediator, and some of the general transcription factors can be isolated from cells as a single complex in the absence of DNA. This led to the speculation that the bulk of the proteins required to initiate transcription might arrive at the promoter in a single preformed complex, rather than in a stepwise manner. The putative preformed complex was named the RNA Pol 11 holoenzyme, after the bacterial enzyme containing the o factor, and thus able to initiate. Despite this parallel in naming, there are essential factors (such as TFIID) that do not associate with the eukaryotic RNA polymerase. It is unclear whether the holoenzyme exists in significant amounts in vivo, compared to separate polymerase and Mediator Complex.

A New Set of Factors Stimulate Pol II Elongation and RNA Proofreading

Once polymerase has initiated transcription, it shifts into the elongation phase, as we have discussed. This transition involves the Pol II enzyme shedding most of its initiation factors — for example, the general transcription factors and Mediator. In their place another set of factors is recruited. Some of these (such as TF11S and hSFT5) are elongation factors—that is, factors that stimulate elongation. Others are required for RNA processing. The enzymes involved in all these processes are, like several of the initiation factors we have discussed, recruited to the C-terminal tail of the large subunit of Pol II, the CTD (Figure 12-18). In

FIGURE 12-18 RNA processing enzymes are recruited by the tail of polymerase. The top part of the figure shows various enzymes involved in RNA processing recruited by die "tail" of polymerase. Different enzymes are recruited depending on the phosphorylation state of the tail, "those enzymes are then transferred to the RNA as they are needed fsee next section in text). The bottom part of the figure illustrates a schematic of the tad, with the sequence of one copy of the heptapeptide repeat shown The positions of serine residues that get phosphorylated are indicated. Phosphorylation of serine at position 5 is associated with recruitment of capping factors, whereas phosphorylation of serine at position 2 is associated with recruitment of splidng factors.

FIGURE 12-18 RNA processing enzymes are recruited by the tail of polymerase. The top part of the figure shows various enzymes involved in RNA processing recruited by die "tail" of polymerase. Different enzymes are recruited depending on the phosphorylation state of the tail, "those enzymes are then transferred to the RNA as they are needed fsee next section in text). The bottom part of the figure illustrates a schematic of the tad, with the sequence of one copy of the heptapeptide repeat shown The positions of serine residues that get phosphorylated are indicated. Phosphorylation of serine at position 5 is associated with recruitment of capping factors, whereas phosphorylation of serine at position 2 is associated with recruitment of splidng factors.

this case, however, the factors favor Ihe phosphurylated forni of the CTD. Thus ph osphory latfqn of the CTD leads I o an exchange of initiation factors for those factors required for elongation and RNA processing.

As is evident from the crystal structure of yeast Pol II, the polymerase CTD lies directly adjacent to the channel through which the newly synthesized RNA exits the enzyme. This, together with its length (it can extend some 800 Á from the body of the enzyme) allows the tail to bind several components of the elongation and processing machinery arid to deliver them to the emerging RNA.

Various proteins are thought to stimulate elongation by Pol II. One of these, the kinase P-TEFb, is recruited to polymerase by transcriptional activators. Once bound to Pol II, this protein phosphorylates the serine residue at position 2 of the CTD repeats as described earlier. That phosphorylation event correlates with elongation. In addition, P-TEFb phosphorylates and thereby activates another protein, called hSPT5f itself an elongation factor. Lastly, TAT-SFl, yet another elongation factor, is recruited by P-TEFb, Thus, P-T£Fb stimulates elongation in three separate ways.

Another factor that does not affect initiation, but stimulates elongation, is TFIIS. This factor stimulates the overall rate of elongation by limiting (he length of time polymerase pauses when it encounters sequences that would otherwise tend to slow the enzyme's progress. It is a feature of polymerase that it does not transcribe through all sequences at a constant rate. Rather, it pauses periodically, sometimes for rather long periods, before resuming transcription. In the presence of TFIIS, the length of time polymerase pauses at any given site is reduced.

TF1IS has another function: it contributes to proofreading by polymerase. We saw at the start of the chapter how polymerases are able, inefficiently, to remove rnisincorporated bases using the active site of the enzyme to perform the reverse reaction to nucleotide incorporation. In addition, TFIIS stimulates an inherent RNAse activity in polymerase (not part of the active site), allowing an alternative approach to remove rnisincorporated bases through local limited RNA degradation. This feature is comparable to the hydrolytic editing we described in the bacterial case stimulated by the Gre factors we discussed there.

Elongating Polymerase Is Associated with a New Set of Protein Factors Required for Various Types of RNA Processing

Once transcribed, eukaryotic RNA has to be processed in various ways before being exported from the nucleus where it can be translated. These processing events include the following: capping of the 5' end of the RNA; splicing; and polyadenylation of the 3' end of the RNA. The most complicated of these is splicing-—the process whereby non-coding introns are removed from RNA to generate the mature mRNA. The mechanisms and regulation of that process and others, such as RNA editing, are the subject of Chapter 13. We consider the other two processes here.

Strikingly, there is an overlap in proteins involved in elongation, and those required for RNA processing. In one case, for example, one elongation factor mentioned above (hSPT5) also recruits aud stimulates the 5' capping enzyme. In another case, elongation factor TAT-SFl recruits components of the splicing machinery. Thus it seems that elongation, termination of transcription, and RNA processing are interconnected — presumably to ensure their proper coordination.

The firsl RNA processing event is capping. This involves the addition of a modified guanine base to the 5' end of the RNA. Specifically, it is a methylated guanine, and it is joined to the RNA transcript by an unusual 5'-5' linkage involving three phosphates (see bottom of Figure 12-19). The 5' cap is created in three enzymatic steps, as detailed in the figure and legend. In the first step, o phosphate group is removed from the 5' of the transcript. Then, the CTP is added. And in the final step, that nucleotide is modified by the addition of a methyl group. The RNA is capped when it is still only some 20 -40 nucleotides long—when the transcription cycle has progressed only to the transition between the initiation and elongation phases. After capping, dephosphorylation of Ser5 within the tail repeats leads to dissociation of the capping machinery, and further phosphorylation (this time of Ser2 nithin the tail repeats) causes recruitment of the machinery needed lor RNA splicing (see Figure 12-18).

The final RNA processing event, polyadenylation of the 3' end of the mRNA, is intimately linked with the termination of transcription (Figure 12-20). Just as with capping and splicing, the polymerase CTD tail is involved in recruiting the enzymes necessary for polyadenylation

FIGURE 12-19 The Structure and formation of the 5* RNA cap- In the first step, the y phosphate at the 5' end of the RNA is removed by an enzyme called RNA triphosphatase (the initiating nucleotide of a transcript initially retains its or, p-, and f-phosphates) in the next step, the enzyme guanylyl transferase catalyzes the nudeaphiiic attack of the resulting terminal p-ptiosphate on the a-phosphoryl group of a molecule of GTR with p- and -y-phosphates of the CTP serving as a pyrophosphate leaving group. Once this linkage is made, tfie newly added guanine and the purine at the or.ginal 5' end of the mRNA are further modified by the addition of methyl groups by methyl transferase The resulting 5' cap structure later recruits the ribosome to the mRNA for translation to begin (see Chapter 14).

guanylyl transferase methyl transferase

7 methyl

3'HO

RNA triphosphatase methyl transferase

RNA triphosphatase

' i guanylyl transferase

3'HO

7 methyl poly-A signal sequence in DNA

poly-A signal sequence in DNA

additional poly-A-binding protein "X
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