The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II). Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes. Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2). Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events. Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association. Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II.

The yeast Paf1 complex (Paf1C: Paf1, Cdc73, Ctr9, Rtf1, and Leo1) is associated with RNA Polymerase II (Pol II) at promoters and coding regions of transcriptionally active genes, but transcript abundance for only a small subset of genes is altered by loss of Paf1. By using conditional and null alleles of PAF1 and microarrays, we determined the identity of both primary and secondary targets of the Paf1C. Neither primary nor secondary Paf1C target promoters were responsive to loss of Paf1. Instead, Paf1 loss altered poly(A) site utilization of primary target genes SDA1 and MAK21, resulting in increased abundance of 3'-extended mRNAs. The 3'-extended MAK21 RNA is sensitive to nonsense-mediated decay (NMD), as revealed by its increased abundance in the absence of Upf1. Therefore, although the Paf1C is associated with Pol II at initiation and during elongation, these critical Paf1-dependent changes in transcript abundance are due to alterations in posttranscriptional processing.

The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3-end formation observed in the absence of Paf1.In Saccharomyces cerevisiae, the Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, is found associated with RNA polymerase II (Pol II) from the promoters to the poly(A) sites of actively transcribed genes (23,31,32). The homologous complex in humans is composed of human Paf1 (hPaf1), hCtr9, hCdc73, and hLeo1 (45,56,60). Although there is an hRtf1 homolog, it does not appear to be part of the hPaf1C. The yeast Paf1C is recruited to Pol II at an as-yetuncharacterized early step in the transcription cycle, subsequent to initiation complex formation (16), which is dependent on the Bur1 kinase (27). The presence of Paf1C is required for the histone H2B monoubiquitylation activity of Rad6; the modified histone H2B subsequently serves as the necessary substrate for H3 lysine 4 (K4) methylation by Set1 and H3 K79 methylation by Dot1 (27,33,54,55). Therefore, the loss of the Paf1C ultimately results in changes in histone modifications.Transcription by Pol II includes a cycle of phosphorylation and dephosphorylation of the C-terminal domain (CTD) of the largest Pol II subunit (reviewed in references 4, 7, 36, 39, 42, and 61). The initiating form of the enzyme is unphosphorylated; initiation and promoter escape are associated with, although not dependent on (21), phosphorylation of serine 5 (Ser5) of the YSPTSPS repeat of the CTD. During elongation, there is additional phosphorylation of serine 2 (Ser2) of the repeat. At, or just after, termination, the CTD is dephosphorylated, resetting Pol II for another transcription cycle. These modifications create unique interaction sites for a large and growing collection of transcriptional and posttranscriptional ...

Our investigations have identified a mechanism by which exogenous production of nitric oxide (NO) induces resistance of Gram-positive and -negative bacteria to aminoglycosides. An NO donor was found to protect Salmonella spp. against structurally diverse classes of aminoglycosides of the 4,6-disubstituted 2-deoxystreptamine group. Likewise, NO generated enzymatically by inducible NO synthase of gamma interferon-primed macrophages protected intracellular Salmonella against the cytotoxicity of gentamicin. NO levels that elicited protection against aminoglycosides repressed Salmonella respiratory activity. NO nitrosylated terminal quinol cytochrome oxidases, without exerting long-lasting inhibition of NADH dehydrogenases of the electron transport chain. The NO-mediated repression of respiratory activity blocked both energy-dependent phases I and II of aminoglycoside uptake but not the initial electrostatic interaction of the drug with the bacterial cell envelope. As seen in Salmonella, the NO-dependent inhibition of the electron transport chain also afforded aminoglycoside resistance to the clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Together, these findings provide evidence for a model in which repression of aerobic respiration by NO fluxes associated with host inflammatory responses can reduce drug uptake, thus promoting resistance to several members of the aminoglycoside family in phylogenetically diverse bacteria.