Upstream Interactions Research Articles

Update of human and mouse forkhead box (FOX) gene families

The forkhead box (FOX) proteins are transcription factors that play complex and important roles in processes from development and organogenesis to regulation of metabolism and the immune system. There are 50 FOX genes in the human genome and 44 in the mouse, divided into 19 subfamilies. All human FOX genes have close mouse orthologues, with one exception: the mouse has a single Foxd4, whereas the human gene has undergone a recent duplication to a total of seven (FOXD4 and FOXD4L1 → FOXD4L6). Evolutionarily ancient family members can be found as far back as the fungi and metazoans. The DNA-binding domain, the forkhead domain, is an example of the winged-helix domain, and is very well conserved across the FOX family and across species, with a few notable exceptions in which divergence has created new functionality. Mutations in FOX genes have been implicated in at least four familial human diseases, and differential expression may play a role in a number of other pathologies -- ranging from metabolic disorders to autoimmunity. Furthermore, FOX genes are differentially expressed in a large number of cancers; their role can be either as an oncogene or tumour suppressor, depending on the family member and cell type. Although some drugs that target FOX gene expression or activity, notably proteasome inhibitors, appear to work well, much more basic research is needed to unlock the complex interplay of upstream and downstream interactions with FOX family transcription factors.

Models of vertical market relations

I contrast various models of vertical contracting that view upstream interactions either through the lenses of bilateral contracting and negotiations or through the lenses of a “market interface” with uniform contractual terms. Existing models contrast starkly in their policy implications, in particular when imperfect horizontal competition, on either the upstream or the downstream level, interacts with differential buyer power. Depending on industry characteristics, different assumptions on contracting may be appropriate. Even though the quest for an all-encompassing modelling framework seems vain, existing models can still be made more flexible, so as to be of greater practical usage.

Alternative Models for Moment Inequalities

Behavioral choice models generate inequalities which, when combined with additional assumptions, can be used as a basis for estimation. This paper considers two sets of such assumptions and uses them in two empirical examples. The second example examines the structure of payments resulting from the upstream interactions in a vertical market. I then mimic the empirical setting for this example in a numerical analysis which computes actual equilibria, examines how their characteristics vary with the market setting, and compares them to the empirical results. The final section uses the numerical results in a Monte Carlo analysis of the robustness of the two approaches to estimation to their underlying assumptions.

Sequence-Dependent Upstream DNA–RNA Polymerase Interactions in the Open Complex with λP R and λP RM Promoters and Implications for the Mechanism of Promoter Interference

Upstream interactions of Escherichia coli RNA polymerase (RNAP) in an open promoter complex (RPo) formed at the P R and P RM promoters of bacteriophage λ have been studied by atomic force microscopy. We demonstrate that the previously described 30-nm DNA compaction observed upon RPo formation at P R [Rivetti, C., Guthold, M. & Bustamante, C. (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J., 18, 4464–4475.] is a consequence of the specific interaction of the RNAP with two AT-rich sequence determinants positioned from − 36 to − 59 and from − 80 to − 100. Likewise, RPos formed at P RM showed a specific contact between RNAP and the upstream DNA sequence. We further demonstrate that this interaction, which results in DNA wrapping against the polymerase surface, is mediated by the C-terminal domains of α-subunits (carboxy-terminal domain). Substitution of these AT-rich sequences with heterologous DNA reduces DNA wrapping but has only a small effect on the activity of the P R promoter. We find, however, that the frequency of DNA templates with both P R and P RM occupied by an RNAP significantly increases upon loss of DNA wrapping. These results suggest that α carboxy-terminal domain interactions with upstream DNA can also play a role in regulating the expression of closely spaced promoters. Finally, a model for a possible mechanism of promoter interference between P R and P RM is proposed.

THE MECHANISM OF UNSTEADY DOWNSTREAM INTERACTIONS OF PREMIXED HYDROGEN-AIR FLAMES

The process of flame annihilation resulting from downstream interactions of premixed hydrogen-air flames is studied using Direct Numerical Simulations (DNS). The process is investigated during unsteady interaction between a vortex pair and a premixed flame kernel in 2D. The annihilation process results from the interactions of the premixed flames on their products' side. The simulations show that the mechanism of extinction during downstream interaction is different from an upstream interaction, which is governed by the sequence of the interactions of the different preheat and reaction layers on both processes and the diffusive transport of heat and mass. In contrast to observations related to upstream interactions, downstream interactions lead to a shift in the equivalence ratio towards the richer conditions with a steady decrease in chemical activity and no radical pool build-up during the stages of extinction. The effect of vortex sizes is qualitatively the same between the different cases considered.

Extending green practices across the supply chain

PurposeThis research aims to extend the “collaborative paradigm” proposed by others in prior research beyond a supply chain's core operations. To date, this paradigm has generated relatively little empirical research on peripheral, non‐core areas such the natural environment. Antecedents (both plant‐level and supply chain characteristics) of green supply chain practices (GSCP) are examined. Among possible antecedents, prior research pointed to supply chain integration – both logistical (tactical level) and technological (strategic level) – as a potentially important determinant of green practices.Design/methodology/approachGreen practices are defined along the two dimensions of environmental collaboration and monitoring. The empirical analysis used data from 84 plants in North America surveyed in 2002. Validity and reliability of scales for new and existing constructs were assessed through factor analysis. Hierarchical linear regression was used to test the hypotheses for the antecedents of GSCP.FindingsTechnological integration with primary suppliers and major customers was positively linked to environmental monitoring and collaboration. For logistical integration, a linkage was found only with environmental monitoring of suppliers. Finally, as the supply base was reduced, the extent of environmental collaboration with primary suppliers increased.Research limitations/implicationsGreater supply chain integration can benefit environment management in operations, and the collaborative paradigm can be extended to this domain. A limitation is that the empirical analysis focused on one industry representing a single echelon.Originality/valueThis is one of the few studies that conceptualize and empirically test GSCP, and consider both and separately upstream and downstream interactions in the supply chain.

Remodeling of the σ 70 Subunit Non-template DNA Strand Contacts During the Final Step of Transcription Initiation

Transcription initiation in bacteria requires melting of approximately 13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the beta subunit lobe I (amino acid residues 186-433) initiates melting of the -10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (+1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by cross-linking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the sigma70 subunit and the non-template strand of the -10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new sigma70-DNA contact as well as stable beta' and beta subunit contacts with the non-template DNA downstream of the -10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that beta lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (beta' clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between sigma70 and the -10 promoter element that might facilitate promoter escape and sigma release.

Sequence-independent upstream DNA–αCTD interactions strongly stimulate Escherichia coli RNA polymerase- lacUV5 promoter association

The C-terminal domains of the two alpha-subunits (alphaCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA-alphaCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA-alphaCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of alphaCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of alphaCTD decreased the composite second-order association rate constant, k(a), of RNAP for the lacUV5 promoter by approximately 10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k(2). Removal of promoter DNA upstream of the -35 element also decreased both k(a) and k(2) approximately 10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The alphaCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with alphaCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the alpha-binding sites. In addition to highlighting the functional importance of nonspecific protein-DNA interactions, these results suggest also that UP element-alphaCTD interactions play an even larger role in transcription initiation than appreciated previously.

A Dominant Negative Gαs Mutant That Prevents Thyroid-stimulating Hormone Receptor Activation of cAMP Production and Inositol 1,4,5-Trisphosphate Turnover: COMPETITION BY DIFFERENT G PROTEINS FOR ACTIVATION BY A COMMON RECEPTOR

A Ser to Asn mutation at position 54 of the alpha subunit of G(s) (designated N54-alpha(s)) was characterized after transient expression of it with various components of the receptor-adenylyl cyclase pathway in COS-1, COS-7, and HEK 293 cells. Previous studies of the N54-alpha(s) mutant revealed that it has a conditional dominant negative phenotype that prevents hormone-stimulated increases in cAMP without interfering with the regulation of basal cAMP levels (Cleator, J. H., Mehta, N. D., Kurtz, D. K., Hildebrandt, J. D. (1999) FEBS Lett. 243, 205-208). Experiments reported here were conducted to localize the mechanism of the dominant negative effect of the mutant. Competition studies conducted with activated alpha(s)* (Q212L) showed that the N54 mutant did not work down-stream by blocking the interaction of endogenous alpha(s) with adenylyl cyclase. The co-expression of wild type or N54-alpha(s) along with the thyroid-stimulating hormone (TSH) receptor and adenylyl cyclase isotypes differing with respect to betagamma stimulation (AC II or AC III) revealed that the phenotype of the mutant is not dependent upon the presence of adenylyl cyclase isoforms regulated by betagamma. These studies ruled out a downstream site of action of the mutant. To investigate an upstream site of action, N54-alpha(s) was co-expressed with either the TSH receptor that activates both alpha(s) and alpha(q) or with the alpha(1B)-adrenergic receptor that activates only alpha(q). N54-alpha(s) failed to inhibit alpha(1B)-adrenergic receptor stimulation of inositol 1,4,5-trisphosphate production but did inhibit TSH stimulation of inositol 1,4,5-trisphosphate. These results show that G(s) and G(q) compete for activation by the TSH receptor. They also indicate that the N54 protein has a dominant negative phenotype by blocking upstream receptor interactions with normal G proteins. This phenotype is different from that seen in analogous mutants of other G protein alpha subunits and suggests that either regulation or protein-protein interactions differ among G protein alpha subunits.

Brain Gene Expression: Genomics and Genetics

Publisher Summary This chapter explains the genomics and genetics of the brain gene expression. Gene expression in the central nervous system (CNS) modulates synaptic efficacy, signal transduction, and numerous other cell functions involved in information processing and cellular homeostasis. Variation in gene expression is influenced by multiple factors, including polymorphic upstream modifier genes, environmental factors, and interactions between genetic and environmental factors. These factors cause gene expression to vary along a continuum. Microarray-based assays of expression measure the steady-state relative abundance of mRNAs. Because transcript abundance is a polygenic trait determined by the action of many other genes, and both internal and external environmental factors complex trait analysis methods can be profitably applied to study the genetic control of gene expression. Complex trait analysis or quantitative trait locus (QTL) mapping involves associations of known genetic markers with phenotypical values in the progeny of a segregating cross. Transcriptome-QTL analysis creates new challenges in multiple testing corrections. By exploiting individual genetic differences, a complex gene regulatory network can be defined, dissected, and perturbed experimentally. It is also possible to locate and identify key genes that generate downstream differences in mRNA abundance. Some of these key modulators of mRNA abundance may prove useful targets for disease prevention and treatment. Building a model of brain function from multipoint, multi-tissue gene expression, and phenomic observation is also described in the chapter.

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

On Cauchy conditions for asymmetric mixed convection boundary layer flows

The fundamental question of how and where does an asymmetric mixed convection boundary layer flow around a heated horizontal circular cylinder begin to develop is raised. We first transform the classical boundary layer equations by using an integral method of Karman–Pohlhausen type and obtain two coupled equations governing the evolutions of the dynamic and thermal boundary layers. Because of its global character, the implemented method allows to bypass the difficulty of downstream–upstream interactions. Cauchy conditions characterizing the starting of the boundary layers are found; they are obtained in a surprisingly simple manner for the limiting cases corresponding to Pr=1, Pr→0 and Pr→∞. Otherwise, these conditions can be found by using a prediction correction algorithm. Some numerical experiments are finally performed in order to illustrate the theory.

Impact of supply chain sensitivity to quality certification on quality management practices and performances

Recent studies on quality management, certification, and supply chain management emphasize that (1) ISO 9000 certification is becoming a must for many companies to compete, and (2) quality is a strategic variable that should be considered and managed not only within the single firm, but also across the supply chain of which the firm is part. Nevertheless, many empirical works on ISO 9000 concentrate on the impact of certification on companies' internal quality systems, whereas they do not pay the same attention to both upstream and downstream interactions across the supply chain, and to their influences on quality management practices and performance. This study examines whether the diffused adoption or knowledge/confidence in the ISO 9000 quality system requirements (i.e. 'sensitivity' to certification) by diverse supply chain members can really influence quality management practices/procedures and operational performance of individual firms in supply chains. These topics are examined through analysis of data gathered by means of a survey based on a sample of 100 Italian certified manufacturing companies. It was found that those firms with the most advanced internal quality systems tend to buy extensively from certified suppliers and to be reliant on the quality level of their deliveries. However, such trust on certified suppliers rarely implies the formalization of medium-long term cooperative programmes. As for performances, the impact of supply chain sensitiveness seems to be significant on punctuality, delivery speed and volume flexibility, while there are no significant effects on quality and cost performance.

DNA replication blockade impairs p53-transactivation.

Tumor-suppressor genes are necessary for maintaining cell growth control. When these genes fail to function properly, deregulated growth—a defining feature of cancer cells—ensues. The p53 tumor suppressor, first described in 1979 as an interacting protein with the transforming large T-antigen of simian virus 40, has emerged as the most commonly altered gene and growth suppressing pathway in human cancer. p53 protein is stabilized in response to a number of stresses, including exposure of cells to DNA damaging agents, hypoxia, or inappropriate expression or activation of a number of cellular or viral oncogenes. The upstream regulatory interactions and downstream events that follow from p53 stabilization provide a complex network of signals ultimately leading to cell cycle arrest, apoptosis, and tumor suppression (1, 2). We know relatively little, however, about the cell cycle dependence of the p53 response, and the role of p53 in the response to replication blockade in S-phase of the cell cycle. Studies reported by Gottifredi et al. (3) in this issue of PNAS shed light on the p53 response in S-phase, differentiate between passage through S-phase and blockade in S-phase in terms of the events downstream of p53 activation, and provide evidence that the transcriptional program downstream of “activated” p53 is drastically attenuated if DNA synthesis is blocked. Interesting questions arise regarding how and why cells stabilize and inhibit p53 during S-phase blockade and parallels can be drawn to the transforming adenovirus E1A protein or the hypoxia inducible factor activating drug desferoxamine, both of which also stabilize p53 while inhibiting its transactivation potential.

Time-resolved vane-rotor-vane interaction in a transonic one-and-a-half stage turbine

This paper describes the effect of both an upstream and a downstream vane on the time-resolved surface pressure field around a high-pressure rotor blade. The geometry of the downstream vane considered is a large low aspect ratio vane located in a swan-necked diffuser duct, similar to those likely to be used in future engine designs. Two test geometries are considered: firstly, a high-pressure turbine stage coupled with a swan-necked diffuser exit duct; secondly, the same high-pressure stage but with a vane located in the downstream duct. Both tests were conducted at engine-representative Mach and Reynolds numbers, and experimental data were acquired using fast response pressure transducers mounted on the mid-height streamline of the high-pressure rotor blades. It is shown that the potential field of the downstream vane causes a relatively large static pressure fluctuation on the late rotor suction surface but that the early suction surface and pressure surface are unaffected by the presence of the vane. In addition to the two main interaction mechanisms (upstream vane-rotor and downstream vane-rotor interactions) a third interaction is identified. This is shown to be caused by the interaction, within the rotor passage, between the flowfield associated with the upstream vane and the potential field associated with the downstream vane. This new interaction mechanism is shown to cause static pressure fluctuations on the late rotor suction surface at a frequency corresponding to the difference in the numbers of upstream and downstream vanes.

A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides −24 to −15 that forms a long-range pairing, termed Alt 1, with the 3′ strand of P2 (P23′) located at the very 3′-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the −24/−15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides −54 to −18, termed P(-1)/L(-1), which sequesters the majority of the −24/−15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme.

Mechanism of ATP-dependent promoter melting by transcription factor IIH.

We show that transcription factor IIH ERCC3 subunit, the DNA helicase responsible for adenosine triphosphate (ATP)-dependent promoter melting during transcription initiation, does not interact with the promoter region that undergoes melting but instead interacts with DNA downstream of this region. We show further that promoter melting does not change protein-DNA interactions upstream of the region that undergoes melting but does change interactions within and downstream of this region. Our results rule out the proposal that IIH functions in promoter melting through a conventional DNA-helicase mechanism. We propose that IIH functions as a molecular wrench: rotating downstream DNA relative to fixed upstream protein-DNA interactions, thereby generating torque on, and melting, the intervening DNA.

Flame Structure Interactions and State Relationships in an Unsteady Partially Premixed Flame

In this investigation our objective is 1 ) to compare the similitude between an unsteady, two-dimensional (axisymmetric), partially premixed e ame and an analogous steady, partially premixed e amelet to determine if state relationshipsintermsofamodie edconservedscalarapplyand2 )toinvestigatee amestructureinteractionsbetween the various reaction zones contained in partially premixed e ames. This comparison is of fundamental importance to theunderstanding and modeling of turbulente amesbecause the axisymmetrice ameinvolvesrelatively complex e ow chemistry interactions resulting from differential diffusion, e ame curvature, and spatiotemporally varying strain rates, whereas the development of state relationships generally assumes negligible differential diffusion effects. A time-dependent, axisymmetric model based on a direct numerical simulation methodology using a relatively detailed CH 4‐airchemical mechanism isemployed to model inverseaxisymmetric partially premixed e ames that are established by introducing a fuel-rich (CH4‐air) annular jet that is sandwiched between an air jet (on the inside)and coe owing air (on theoutside ). The e ame consists of distinct layers that include 1 ) an inner layer (PF) in which methane and O 2 consumption occur and 2 ) an oxidation layer (NF). The broadened inner premixed e ame is synergistically coupled with an oxidation layer, and the upstream region of the nonpremixed e ame is contained downstream of the premixed e ame. The signie cant hydrocarbon chemistry occurs almost solely in the PF where fuel and radical consumption produce CO and H 2, which are then oxidized to form CO 2 and H2O in the NF. The nonpremixed e ame provides radicals to accelerate the upstream region of the premixed e ame. Comparison with an analogous e amelet reveals that transport effects in the axisymmetric e ame are signie cant on the rich side. The coannulare amescalar proe lesshow regions of both frozen e ow and burning. Thescalar distributions in thee ame, when compared with analogous e amelet proe les, indicate that upstream interactions occur 1 ) in the rich region with the consequence of enhanced heat release, 2 ) at the nonpremixed interface leading to higher heat release through H2 and CO oxidation, and 3 ) in the lean region where methane consumption occurs despite the local equivalence ratios being well below the lean e ammability limit. The synergistic interactions between the inner and outer layers lead to the formation of complex composite e ames.

NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.

The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.

Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter.

The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of pRM's ATR. We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM. However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions. The possible involvement of the alpha subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.