The Protective Effect of Virus Capsids on RNA and DNA Virus Genomes in Wastewater.

Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the T90 for MS2 (+)ssRNA increased by 424× and the T90 for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.

MRB10130 is a RESC assembly factor that promotes kinetoplastid RNA editing initiation and progression.

Uridine insertion deletion editing in kinetoplastid protozoa requires a complex machinery, a primary component of which is the RNA editing substrate binding complex (RESC). RESC contains two modules termed GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex), although how interactions between these modules and their mRNA and gRNA binding partners are controlled is not well understood. Here, we demonstrate that the ARM/HEAT repeat containing RESC protein, MRB10130, controls REMC association with mRNA- and gRNA-loaded GRBC. High-throughput sequencing analyses show that MRB10130 functions in both initiation and 3' to 5' progression of editing through gRNA-defined domains. Editing intermediates that accumulate upon MRB10130 depletion significantly intersect those in cells depleted of another RESC organizer, MRB7260, but are distinct from those in cells depleted of specific REMC proteins. We present a model in which MRB10130 coordinates numerous protein-protein and protein-RNA interactions during editing progression.

Marked for translation: long A/U tails as an interface between completion of RNA editing and ribosome recruitment.

The mechanism by which ribosomes select translatable mRNAs from the complex mixture of incompletely edited mRNAs in trypanosome mitochondria has remained a mystery. In this issue of Molecular Cell, Aphasizheva and colleagues (Aphasizheva et al., 2011) reveal a role for long 3' A/U tails in signaling ribosome recruitment to a fully edited, translatable mRNA.

Structures of the T. brucei kRNA editing factor MRB1590 reveal unique RNA-binding pore motif contained within an ABC-ATPase fold.

Kinetoplastid RNA (kRNA) editing is a process that creates translatable mitochondrial mRNA transcripts from cryptogene encoded RNAs and is unique for kinetoplastids, such as Trypanosoma brucei. In addition to the catalytic 20S editosome, multiple accessory proteins are required for this conversion. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. MRB1 consists of six core proteins but makes dynamic interactions with additional accessory proteins. Here we describe the characterization of one such factor, the 72 kDa MRB1590 protein. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). Structural studies show that MRB1590 is dimeric and contains a central ABC-ATPase fold embedded between novel N- and C-terminal regions. The N-terminal domains combine to create a basic pore and biochemical studies indicate residues in this region participate in RNA binding. Structures capturing distinct MRB1590 conformations reveal that the RNA binding pore adopts closed and open states, with the latter able to accommodate RNA. Based on these findings, implications for MRB1590 function are discussed.

An arginine-glycine-rich RNA binding protein impacts the abundance of specific mRNAs in the mitochondria of Trypanosoma brucei.

In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo, inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei. TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

Architecture of the trypanosome RNA editing accessory complex, MRB1.

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.

MRB3010 is a core component of the MRB1 complex that facilitates an early step of the kinetoplastid RNA editing process.

Gene expression in the mitochondria of the kinetoplastid parasite Trypanosoma brucei is regulated primarily post-transcriptionally at the stages of RNA processing, editing, and turnover. The mitochondrial RNA-binding complex 1 (MRB1) is a recently identified multiprotein complex containing components with distinct functions during different aspects of RNA metabolism, such as guide RNA (gRNA) and mRNA turnover, precursor transcript processing, and RNA editing. In this study we examined the function of the MRB1 protein, Tb927.5.3010, which we term MRB3010. We show that MRB3010 is essential for growth of both procyclic form and bloodstream form life-cycle stages of T. brucei. Down-regulation of MRB3010 by RNAi leads to a dramatic inhibition of RNA editing, yet its depletion does not impact total gRNA levels. Rather, it appears to affect the editing process at an early stage, as indicated by the accumulation of pre-edited and small partially edited RNAs. MRB3010 is present in large (>20S) complexes and exhibits both RNA-dependent and RNA-independent interactions with other MRB1 complex proteins. Comparison of proteins isolated with MRB3010 tagged at its endogenous locus to those reported from other MRB1 complex purifications strongly suggests the presence of an MRB1 "core" complex containing five to six proteins, including MRB3010. Together, these data further our understanding of the function and composition of the imprecisely defined MRB1 complex.

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites.

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.

gRNA/pre-mRNA annealing and RNA chaperone activities of RBP16.

Editing in trypanosomes involves the addition or deletion of uridines at specific sites to produce translatable mitochondrial mRNAs. RBP16 is an accessory factor from Trypanosoma brucei that affects mitochondrial RNA editing in vivo and also stimulates editing in vitro. We report here experiments aimed at elucidating the biochemical activities of RBP16 involved in modulating RNA editing. In vitro RNA annealing assays demonstrate that RBP16 significantly stimulates the annealing of gRNAs to cognate pre-mRNAs. In addition, RBP16 also facilitates hybridization of partially complementary RNAs unrelated to the editing process. The RNA annealing activity of RBP16 is independent of its high-affinity binding to gRNA oligo(U) tails, consistent with the previously reported in vitro editing stimulatory properties of the protein. In vivo studies expressing recombinant RBP16 in mutant Escherichia coli strains demonstrate that RBP16 is an RNA chaperone and that in addition to RNA annealing activity, it contains RNA unwinding activity. Our data suggest that the mechanism by which RBP16 facilitates RNA editing involves its capacity to modulate RNA secondary structure and promote gRNA/pre-mRNA annealing.

One- and two-stage anaerobic co-digestion of cucumber waste and sewage sludge.

The demand for uniformly sized and shaped produce that are aesthetically pleasing results in significant food waste throughout the world. Cucumber waste is a major agricultural waste product in a number of countries, especially areas with high pickle production. Opportunity exists for wastewater treatment plants containing anaerobic digesters to utilize cucumber agricultural and industrial waste for biogas production. The biomethane potential of cucumber waste as a substrate for co-digestion with sewage sludge was assessed. The impact of long-term co-digestion of cucumber was then evaluated using mesophilic continuously stirred tank reactors (CSTRs), in both single- and two-stage anaerobic co-digestion with sewage sludge. Ground cucumber waste was added to sewage sludge at 8% of the volume (4.5-4.6% of the organic load) and CSTRs were maintained for five hydraulic retention times (HRTs). One-stage co-digestion of cucumber waste produced comparable gas levels as CSTRs without cucumbers (averaging 219 and 221 m3/kgVS/h, respectively) after two HRTs. The two-stage cucumber co-digestion CSTR averaged 64% higher specific gas than the control and single-stage digester, although the volumetric gas produced was lower (averaging 152 m3/kgVS/h) likely due to gas loss in the first stage resulting in a lower organic load rate. After four HRTs, relative methanogen content showed dramatic differences in levels of hydrogenotrophic methanogens for the two-stage digester, while the one-stage digester containing cucumber waste showed minor differences relative to the control. Cucumber waste co-digestion with sewage sludge is effective although numerous conditions could be utilized to optimize gas production.

In-situ extraction and impregnation of black walnut husk into polyethylene film using supercritical carbon dioxide with an ethanol modifier.

Walnuts are commonly cultivated for their kernel, which is a rich source of antioxidant phenolic compounds. The husk likewise contains antioxidant and antimicrobial compounds, but is typically discarded without further processing. Antioxidant compounds are useful in creating active packaging films, but typically decompose at melt extrusion temperatures in polymer processing. Due to carbon dioxide's low critical point and ability to swell polymer films, supercritical carbon dioxide may be used to impregnate phenolic compounds into polymers. For this study, a novel technique is used to simultaneously produce walnut husk extracts and impregnate the extract into polymer films in the same batch extractor using supercritical carbon dioxide with a 15 wt-% ethanol modifier at 60°C at 19.4 MPa. The effect of varying the loading of walnut husk in the extractor upon impregnation mass was evaluated with the impregnation mass of the film increasing with walnut husk loading. It was determined by FTIR, as well as the reduction of the protein cytochrome c, that antioxidant compounds may be extracted from walnut husks and impregnated into low-density polyethylene film (LDPE) by this technique.

Genetic interactions between TFIIF and TFIIS.

The eukaryotic transcript elongation factor TFIIS is encoded by a nonessential gene, PPR2, in Saccharomyces cerevisiae. Disruptions of PPR2 are lethal in conjunction with a disruption in the nonessential gene TAF14/TFG3. While investigating which of the Taf14p-containing complexes may be responsible for the synthetic lethality between ppr2Delta and taf14Delta, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14Delta ppr2Delta cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2Delta and taf14Delta strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF.

Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II.

Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

Dual core processing: MRB1 is an emerging kinetoplast RNA editing complex.

Our understanding of kinetoplastid RNA (kRNA) editing has centered on this paradigm: guide RNAs (gRNAs) provide a blueprint for uridine insertion/deletion into mitochondrial mRNAs by the RNA editing core complex (RECC). The characterization of constituent subunits of the mitochondrial RNA-binding complex 1 (MRB1) implies that it too is vital to the editing process. The recently elucidated MRB1 architecture will be instrumental in putting functional data from individual subunits into context. Our model depicts two functions for MRB1: mediating multi-round kRNA editing by coordinating the exchange of multiple gRNAs required by RECC to edit lengthy regions of mRNAs, and then linking kRNA editing with other RNA processing events.

A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1) complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA) binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function.

Distinct and overlapping functions of MRP1/2 and RBP16 in mitochondrial RNA metabolism.

Mitochondrial RNA metabolism in Trypanosoma brucei is a complex process involving both extensive RNA editing and control of RNA stability. MRP1/2 and RBP16 are two factors that have been implicated in regulating the editing and stability of specific mRNAs. These two factors exhibit similar nonspecific RNA binding and RNA-annealing activities, suggesting that some of their actions may have been previously masked by functional redundancy. Here, we examine the functional interaction of MRP1/2 and RBP16 by separate and simultaneous RNA interference and by overexpressing RBP16 in an MRP1/2-depleted background. Simultaneous depletion of these factors resulted in synthetic lethality in procyclic trypanosomes. Analysis of mitochondrial RNAs in procyclic cells revealed distinct functions for MRP1/2 and RBP16 toward edited apocytochrome b mRNA, redundant functions in stabilization of edited ATPase subunit 6 and cytochrome oxidase subunit 3 mRNAs, and concentration-dependent positive and negative functions for RBP16 toward edited RPS12 mRNAs. While simultaneous MRP1/2-RBP16 depletion had no effect on the growth of bloodstream form cells, massive adverse effects on the levels of almost all mitochondrial RNAs were observed. These studies greatly expand our knowledge regarding the functions of MRP1/2 and RBP16 and suggest that both RNA-specific and life cycle stage-specific factors impact MRP1/2 and RBP16 functions.

TbRGG2, an essential RNA editing accessory factor in two Trypanosoma brucei life cycle stages.

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 down-regulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.

Functions of Saccharomyces cerevisiae TFIIF during transcription start site utilization.

Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of Saccharomyces cerevisiae transcription factor IIF (TFIIF) can cause upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes. In this study, we report the results from multiple biochemical assays analyzing the activities of wild-type yeast TFIIF and the TFIIF Tfg1 mutant containing the E346A substitution (Tfg1-E346A). We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds and dramatically stabilizes a short RNA-DNA hybrid in the RNA polymerase II (RNAPII) active center and, importantly, that the Tfg1-E346A substitution coordinately enhances early bond formation and the processivity of early elongation in vitro. These results are discussed within a proposed model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation.

Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF.

Previous studies have shown that transcription factors IIB (TFIIB), IIF (TFIIF), and RNA polymerase II (RNAPII) play important roles in determining the position of mRNA 5'-ends in the yeast Saccharomyces cerevisiae. Yeast strains containing a deletion of the small, nonessential Rpb9 subunit of RNAPII exhibit an upstream shift in the positions of mRNA 5'-ends, whereas mutation of the large subunit of yeast TFIIF (Tfg1) can suppress downstream shifts that are conferred by mutations in TFIIB. In this study, we report an approach for the production of functional recombinant yeast holo-TFIIF (Tfg1-Tfg2 complex) and use of the recombinant protein in both reconstituted transcription assays and gel mobility shifts in order to investigate the biochemical alterations associated with the deltaRpb9 polymerase. The results demonstrated that upstream shifts in the positions of mRNA 5'-ends could be conferred by the deltaRpb9 RNAPII in transcription reactions reconstituted with highly purified yeast general transcription factors and, importantly, that these shifts are associated with an impaired interaction between the DeltaRpb9 polymerase and TFIIF. Potential mechanisms by which an altered interaction between the DeltaRpb9 RNAPII and TFIIF confers an upstream shift in the positions of mRNA 5'-ends are discussed.