These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.

The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3' end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3' end, then the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential checkpoint in a manner that compromises transcriptional fidelity. In contrast, no effects of Rpb9 on NTP selectivity were observed.

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Rpb9 is a conserved RNA polymerase II (pol II) subunit, the absence of which confers alterations to pol II enzymatic properties and transcription fidelity. It has been suggested previously that Rpb9 affects mobility of the trigger loop (TL), a structural element of Rpb1 that moves in and out of the active site with each elongation cycle. However, a biochemical mechanism for this effect has not been defined. We find that the mushroom toxin ␣-amanitin, which inhibits TL mobility, suppresses the effect of Rpb9 on NTP misincorporation, consistent with a role for Rpb9 in this process. Furthermore, we have identified missense alleles of RPB9 in yeast that suppress the severe growth defect caused by rpb1-G730D, a substitution within Rpb1 ␣-helix 21 (␣21). These alleles suggest a model in which Rpb9 indirectly affects TL mobility by anchoring the position of ␣21, with which the TL directly interacts during opening and closing. Amino acid substitutions in Rpb9 or Rpb1 that disrupt proposed anchoring interactions resulted in phenotypes shared by rpb9⌬ strains, including increased elongation rate in vitro. Combinations of rpb9⌬ with the fast rpb1 alleles that we identified did not result in significantly faster in vitro misincorporation rates than those resulting from rpb9⌬ alone, and this epistasis is consistent with the idea that defects caused by the rpb1 alleles are related mechanistically to the defects caused by rpb9⌬. We conclude that Rpb9 supports intra-pol II interactions that modulate TL function and thus pol II enzymatic properties.Rpb9 is a small (122 amino acids in Saccharomyces cerevisiae) subunit of RNA polymerase II (pol II) 2 that is highly conserved among eukaryotes (1, 2). It is composed of two zinc ribbon domains characterized by the zinc-chelating motif (CX 2 CX n CX 2 C). Although Rpb9 is not essential for growth in yeast, rpb9 null mutants have a number of phenotypes, including slow growth (2), hypersensitivity to several drugs (2-5), upstream shifts in transcription start site (2, 6), defects in transcription-coupled repair that result in enhanced sensitivity to UV light (7), and decreased transcriptional fidelity in vivo (4,8) and in vitro (9, 10).The fidelity defect of pol II that is lacking Rpb9 (pol II⌬9) has been examined in several studies. Decreased selectivity for correct (complementary to the DNA template) versus incorrect NTP substrates has been observed (10). However, our prior experiments have not provided evidence for a selectivity defect, and, in our hands, the decreased fidelity of pol II⌬9 appears to be the result of inefficient proofreading caused by an increased rate of RNA extension from a mismatched 3Ј-end combined with a decreased efficiency of TFIIS-mediated mismatch removal (9). A role for Rpb9 in fidelity is consistent with reported roles for the homologous subunits in RNA polymerases I and III (A12.2 and C11, respectively) (11, 12) as well as the related archaeal protein TFS (13, 14). However, the role that Rpb9 plays in pol II fidelity is probably distinct from these homolo...