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PubMed1995

Changes in ultrasonic Doppler backscattered power downstream of concentric and eccentric stenoses under pulsatile flow.

The main objective of the present work was to investigate, under pulsatile flow, the patterns of variation of the Doppler power backscattered by blood and Sephadex particles upstream and downstream of concentric and eccentric stenoses ranging from 47% to 91% area reduction. Doppler measurements were performed at 5 diameters upstream and 5, 10, 15 and 20 diameters downstream of the constriction. For the concentric 75% and 85%, and the eccentric 79% and 91% area reduction stenoses, a progressive increase of the power backscattered by red cell suspensions at 40% hematocrit was measured downstream of the narrowing. The maximal power usually occurred around 10 diameters after the stenosis and dropped further downstream. In addition to the increase in the power, a cyclic variation of the backscattered intensity was observed within the flow cycle. For the concentric 52% and eccentric 47% area reduction stenoses, no variation of the Doppler power was measured during flow acceleration and deceleration for all recording sites. A coefficient of correlation of 0.82 was measured between the percentage of area reduction and the ratio of the Doppler mean power at 10 diameters downstream to that at 5 diameters upstream of the stenoses. Using Sephadex particles at low concentration, no increase of the Doppler power was found downstream of the 85% and 91% area reduction stenoses. The possible link between the intensity of turbulence and the power backscattered by blood is discussed along with the influence of the correlation between the scattering particles, under turbulent flow.

Acceleration↗
PubMed2004

Central administration of corticotropin-releasing hormone alters downstream movement in an artificial stream in juvenile chinook salmon (Oncorhynchus tshawytscha).

We evaluated the effect of corticotropin-releasing hormone (CRH) on spatial distribution and downstream movement in an artificial stream in juvenile Chinook salmon (Oncorhynchus tshawytscha) during the period when the fish were able to tolerate seawater. An intracerebroventricular (ICV) injection of CRH (500 ng) to hatchery fish significantly increased the proportion of fish that were distributed downstream of a mid-stream release site. A second group of hatchery fish were given ICV injections of saline (control) or CRH (500 ng) and released near the top of the stream. The time taken to enter a trap at the lower end of the stream was recorded. In all cases the groups given CRH had a higher proportion of fish that did not enter the trap within 60 min of release. However, in those fish that did enter the trap, treatment with CRH increased the speed of downstream movement to this point relative to control fish. Wild sub-yearling Chinook salmon were captured during their downstream migration to the estuary and given ICV injections of saline or CRH (500 ng) either 2, 3, or 7 days after transport from the river. As with hatchery fish, a significantly higher proportion of wild fish that were administered CRH did not enter the trap at the lower end of the stream. The mean time of passage for control fish decreased on each successive day (day 2 > day 3 > day 7). In contrast, the mean passage time of the wild fish that were given CRH was relatively constant through time, and was only significantly faster than control fish on day 2. The current study provides evidence that CRH alters the downstream movement of juvenile Chinook in a simulated stream environment, and produces behavioral effects similar to those of juvenile salmonids that are stressed during their downstream migration.

Animal Migration↗
PubMed2006

Effects of growth hormone and cortisol on the downstream migratory behavior in masu salmon, Oncorhynchus masou.

The effects of ovine growth hormone (oGH) and cortisol on downstream migratory behavior in yearling (1(+)) smolts and underyearling (0(+)) parr of masu salmon, Oncorhynchus masou, were examined during the downstream migratory period in spring using artificial raceways. In May, each of 22 1(+) smolts and 0(+) parr were implanted with cholesterol pellets containing 250 microg of oGH and/or 2 mg of cortisol. Their downstream migratory behavior was subsequently observed in artificial raceways, along with control groups 4-23 days after implantation. In 1(+) smolts, the frequency of downstream migratory behavior was 23%, 18%, 72%, and 82% in the control, oGH, cortisol, and oGH+cortisol-treated groups, respectively. The frequency was significantly higher in the cortisol and oGH+cortisol-treated groups than in the control and oGH-treated groups. In 0(+) parr, the frequency of downstream migratory behavior in the cortisol (82%) and cortisol+oGH-treated (90%) groups was significantly higher than in the control (18%) and oGH-treated (0%) groups. These results indicate that cortisol is an important endocrine factor inducing downstream migratory behavior in both 1(+) smolt and 0(+) parr of masu salmon.

Age Factors↗
PubMed2001

The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs.

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.

Base Sequence↗
PubMed1990

A downstream initiation element required for efficient TATA box binding and in vitro function of TFIID.

The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells. In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint. One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site. We have now shown by transfection that both elements are required for efficient transcription in cultured cells. A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator. Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro. In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region. The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription. These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

Base Sequence↗
PubMed1995

Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region.

Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF-I gene transcription associated with diabetes mellitus.

Animals↗
PubMed2000

Downstream promoter sequences facilitate the formation of a specific transcription factor IID-promoter complex topology required for efficient transcription from the megalin/low density lipoprotein receptor-related protein 2 promoter.

Megalin/low density lipoprotein receptor-related protein 2 (LRP-2) is an endocytic receptor expressed in highly specialized cell types such as parathyroid cells and epithelia of the kidney. Previous experiments identified a nonconsensus TATA element, with the sequence TAGAAAA, as crucial for accurate and efficient transcription from the LRP-2 promoter. Here we show that, in addition to the TAGA element, promoter sequences downstream of the transcription start site contribute significantly to transcription both in vitro and in transfected cells. Deletion and point mutational analyses reveal that the promoter region located between positions +5 and +11 (sequence TTTTGGC) is of particular importance. Complementation experiments in nuclear extracts lacking transcription factor IID (TFIID) activity show that TATA-binding protein-associated factors of TFIID are essential for the function of LRP-2 downstream promoter sequences. Interestingly, DNase I footprinting studies show that the downstream region between positions +5 and +11 does not significantly affect overall TFIID affinity to the promoter but that it profoundly affects the topology of the TFIID x promoter complex not only downstream of the transcription start site, but in particular in the TATA box region. Our observations suggest a model for a novel downstream sequence function, in which TATA-binding protein-associated factor-promoter interactions downstream of the transcription start site modulate TFIID-DNA interactions in the TATA box region.

Base Sequence↗
PubMed1996

Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters.

We describe the identification and characterization of a conserved downstream basal promoter element that is present in a subset of Drosophila TATA-box-deficient (TATA-less) promoters by using purified, epitope-tagged TFIID complex (eTFIID) from embryos of transgenic Drosophila. DNase I footprinting of the binding of eTFIID to TATA-less promoters revealed that the factor protected a region that extended from the initiation site sequence (about +1) to approximately 35 nucleotides downstream of the RNA start site. In contrast, there was no apparent upstream DNase I protection or hypersensitivity induced by eTFIID in the -25 to -30 region at which TATA motifs are typically located. Further studies revealed a conserved sequence motif, (A/G)G(A/T)CGTG, termed the downstream promoter element (DPE), which is located approximately 30 nucleotides downstream of the RNA start site of many TATA-less promoters. DNase I footprinting and in vitro transcription experiments revealed that a DPE in its normal downstream location is necessary for transcription of DPE-containing TATA-less promoters and can compensate for the disruption of an upstream TATA box of a TATA-containing promoter. Moreover, a systematic mutational analysis of DNA sequences that encompass the DPE confirmed the importance of the consensus DPE sequence motif for basal transcription and further supports the postulate that the DPE is a distinct, downstream basal promoter element. These results suggest that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters.

Animals↗
PubMed2004

Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems.

The transcriptional factor DREB/CBF (dehydration-responsive element/C-repeat-binding) specifically interacts with the dehydration-responsive element (DRE)/C-repeat (CRT) cis-acting element (A/GCCGAC) and controls the expression of many stress-inducible genes in Arabidopsis. Transgenic plants overexpressing DREB1A showed activated expression of many stress-inducible genes and improved tolerance to not only drought, salinity, and freezing but also growth retardation. We searched for downstream genes in transgenic plants overexpressing DREB1A using the full-length cDNA microarray and Affymetrix GeneChip array. We confirmed candidate genes selected by array analyses using RNA gel blot and identified 38 genes as the DREB1A downstream genes, including 20 unreported new downstream genes. Many of the products of these genes were proteins known to function against stress and were probably responsible for the stress tolerance of the transgenic plants. The downstream genes also included genes for protein factors involved in further regulation of signal transduction and gene expression in response to stress. The identified genes were classified into direct downstream genes of DREB1A and the others based on their expression patterns in response to cold stress. We also searched for conserved sequences in the promoter regions of the direct downstream genes and found A/GCCGACNT in their promoter regions from -51 to -450 as a consensus DRE. The recombinant DREB1A protein bound to A/GCCGACNT more efficiently than to A/GCCGACNA/G/C.

Arabidopsis↗
PubMed2001

Downstream sequences influence the choice between a naturally occurring noncanonical and closely positioned upstream canonical heptameric fusion motif during bovine coronavirus subgenomic mRNA synthesis.

Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5' and 3' coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (-17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5' end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.

Animals↗
PubMed2003

The burden of prevention: downstream consequences of Pap smear testing in the elderly.

CONTEXT: Although cervical cancer is an unusual cause of death among women 65 and older, most elderly women in the US report continuing to undergo periodic Pap smear screening. OBJECTIVE: To describe the incidence of Pap smears and downstream testing among elderly women. SETTING: Claims-based analysis of female Medicare enrollees age 65 and older. METHODS: Using three years of Medicare Part B 5% Files (1995-1997), we differentiated between women undergoing screening Pap smears and those undergoing Pap smears for surveillance of previous abnormalities or Pap smear follow-up. We determined the proportion of elderly women undergoing Pap smear testing and rates of downstream testing and procedures after an initial Pap smear. RESULTS: Four million female Medicare beneficiaries over 65 years underwent Pap smear testing between 1995 and 1997, representing 25% of the eligible population. After adjusting for underbilling for Pap smears under Medicare, 43% of women over 65 are estimated to have undergone Pap smear testing during the 3-year period. The large majority (90%) of Pap smears were for screening, while 10% were done for surveillance or follow-up. For every 1000 women with a screening Pap smear, 39 had at least one downstream intervention within eight months of the initial Pap smear, including seven women who underwent colposcopy and two women who had other surgical procedures. Rates of downstream interventions were considerably higher for women undergoing Pap smear follow-up (302 per 1000 with at least one downstream intervention), and surveillance of previous abnormalities (209 per 1000 with a downstream intervention). CONCLUSION: Cervical cancer screening is widespread among elderly American women, and follow-up testing is not uncommon, particularly among the ten percent of women who appear to be in a cycle of repeated testing. This substantial volume of testing occurs despite the rarity of cervical cancer deaths and unknown benefits of screening in this age group.

Aged↗
PubMed1990

Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences.

Escherichia coli RNA polymerase pauses immediately after transcription of certain sequences that can form stable secondary structures in the nascent RNA transcript; pausing appears to be essential for several types of bacterial transcription attenuation mechanisms. Because base changes that weaken the RNA secondary structures reduce the half-life of pausing by RNA polymerase, nascent transcript RNA hairpins are thought to cause pausing at these sites. We show here that, for the well characterized trpL pause site, the determinants of transcription pausing are not limited to the RNA hairpin, but include the not-yet-transcribed sequence of DNA immediately downstream from the pause site. We show that this effect extends to bases up to fourteen nucleotides downstream from the pause site, that placement of a oligo(dT) tract in the nontranscribed strand in this region does not convert the pause site to a termination site, and that shifting the position of pausing by one nucleotide downstream almost eliminates pausing. From an analysis of many variants of this downstream sequence, we argue that the effect of downstream sequence is not related simply to its GC content. We suggest that these effects are mediated by altered interactions between RNA polymerase and the DNA template downstream from the enzyme's active site.

Base Sequence↗
PubMed2005

Population structure and dynamics of Gammarus fossarum (Amphipoda) upstream and downstream from effluents of sewage treatment plants.

Two streams in Germany (Körsch and Lockwitzbach), each with two sampling sites above and below a sewage treatment plant (STP) discharging effluent, were investigated. Sampling sites were characterized, and exposure monitoring for chemicals with known or assumed endocrine disrupting potential was carried out. Both the population structure and the population dynamics of Gammarus fossarum were examined. The physicochemical parameters measured at the sampling sites of the Lockwitzbach and Körsch streams were found not to reach levels having an acute toxic impact on the development of gammarids. The calculated estrogenic potential in the stream water was 22- to 35-fold higher at the downstream site of the Körsch compared with the other sampling sites, mainly because of the concentrations of 17alpha-ethinylestradiol on two sampling dates. At both streams, an influence of the respective STP effluent on the sex ratio of G. fossarum was not observed. Moreover, intersexuality was not induced by these effluents. Differences in the structure and dynamics of G. fossarum populations were more pronounced at the Körsch than at the Lockwitzbach. At the downstream sampling site at the Körsch, gammarids reached their highest abundances. Particularly at the downstream sampling site of the Körsch, the proportion of breeding female gammarids and the proportion of juvenile gammarids in the smallest body length class were decreased compared with upstream. Adult gammarids were larger from the Lockwitzbach downstream site, but they were smaller from the Körsch downstream site compared with the respective upstream site. At the Körsch, the earlier onset of the autumnal reproductive resting period could be caused by the influence of the STP effluent.

Acanthocephala↗
PubMed1983

Immunoglobulin gene transcription is activated by downstream sequence elements.

To examine the DNA sequences involved in regulation of kappa immunoglobulin gene expression, an assay was developed whereby transcription of a cloned, rearranged kappa gene could be detected following its transfection into antibody-secreting mouse myeloma cells. In a transient expression assay, RNA transcripts initiating 20 to 25 bp before the AUG translation initiator of the kappa gene were readily detected. The constant region (C kappa) and part of the J kappa-C kappa intron was then deleted from the rearranged gene and a downstream SV40 polyadenylylation signal was provided. No transcription was detected from the deleted gene upon transfection into myeloma cells, suggesting that after rearrangement the kappa variable region promoter is activated by sequences more than 2.6 kb downstream of J kappa. The rearranged kappa gene was also transfected into a nonlymphoid line of monkey cells and RNA transcripts were detected. Transcription in these cells, however, was at a relatively lower level than in myeloma cells, did not depend on the presence of downstream sequences, and initiated from a site about 10 bp downstream of the myeloma site. These experiments define a sequence downstream of the promoter that is necessary for accurate transcription of the kappa chain gene.

Animals↗
PubMed1993

Initiator sequences direct downstream promoter binding by human transcription factor IID.

Whereas the human transcription factor IID generally interacts with only the TATA box element on most class II gene promoters, on certain promoters (e.g., the adenovirus 2 major late promoter) TFIID protects DNA sequences up to +35 base pairs downstream of the start site of transcription from DNase I cleavage. In this study, we show that Ad2 MLP sequences from -3 to +5 were sufficient to direct downstream promoter binding by TFIID when introduced into the human hsp70 gene promoter. These sequences correspond to the initiator transcription control element. Initiator mutations resulting in a loss of downstream binding demonstrated a diminished transcription efficiency in vitro. Likewise, initiator-dependent transcription stimulation required TFIID fractions capable of downstream promoter binding. Given the recent findings that immunopurified, human TFIID exhibited downstream promoter binding on the Ad2 MLP (Zhou, Q., Lieberman, P.M., Boyer, T.G. and Berk, A.J. (1992) Genes Dev. 6, 1964-1974), our data suggest that human TFIID can recognize the initiator element and that this interaction is required for maximally efficient transcription initiation.

Base Sequence↗
PubMed2005

The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1.

ISP-1 inhibits de novo sphingolipid biosynthesis and induces growth defects in both mammals and yeast (Saccharomyces cerevisiae). In our previous study, YPK1/SLI2 was identified as one of multicopy suppressor genes for ISP-1 in yeast. Ypk1 is proposed to be a downstream serine/threonine kinase of the sphingolipid signaling pathway in yeast. Other than resistance against ISP-1, Ypk1 is involved in at least two downstream events, namely cell growth and endocytosis. In this study, the effect of mutants of Ypk1 on these three downstream events was investigated. Among Ypk1 mutants, no 'kinase-dead' mutants complemented the defects in any of these three downstream events in the ypk1 null strain. One of the hydrophobic motif phosphorylation-deficient mutants of Ypk1, Ypk1(T662A) had the moderate kinase activity compared with the wild-type Ypk1. Ypk1(T662A) and the wild-type Ypk1 completely restored the slow-growth phenotype and fluid-phase endocytosis defect of the ypk1 null strain. However, unlike the wild-type Ypk1, Ypk1(T662A) lost the ability for the recovery of the ISP-1 resistance in the ypk1 null strain. Furthermore, the expression of Ypk1(T662A) in the wild-type strain showed a dominant-negative effect on the ISP-1-resistance activity. On the other hand, the cell growth revertant of the ypk1 null strain still showed the hypersensitive phenotype to ISP-1. These data suggest that the ISP-1-resistance pathway is under the regulation of the hydrophobic motif phosphorylation and is separated from the other pathways downstream of Ypk1.

Amino Acid Motifs↗
PubMed2006

An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency.

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.

Amino Acid Motifs↗
PubMed2005

Dynamic error correction and regulation of downstream bubble opening by human RNA polymerase II.

The nucleotide triphosphate (NTP)-driven translocation hypothesis posits that NTP substrates bind to templated DNA sites prior to translocation into the active site. By using millisecond phase kinetics, we demonstrate this prediction in three different ways. First, we show that, in the presence of the translocation blocker alpha-amanitin, NTPs (but not deoxynucleotide triphosphate [dNTPs]) templated at downstream sites (i + 2 and i + 3) dislodge an active site (i + 1) NTP, which was otherwise fated to complete bond synthesis. Second, we show that NTPs templated at i + 2 and/or i + 3 downstream sites suppress misincorporation errors. Third, we show that NTPs templated at downstream sites stabilize the posttranslocated elongation complex at a stall position. Therefore, at least two NTP substrates pair to DNA templated sites downstream of the active site. These results demonstrate the mechanisms of NTP loading and transcriptional efficiency and fidelity for human RNA polymerase II and indicate regulation of downstream bubble opening by NTPs.

Catalytic Domain↗