Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase1

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [Km] = 29 μm when UMP is the other substrate and Km = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 μm) and CMP (Km = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P1, P5-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5′-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the ‘AU-box’, just upstream of the SD sequences. To clarify the role of 5′-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 –38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the –38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5′-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5′-UTR and possible roles of the AU-box motif and the SD sequence.

Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog's sacculus.

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, we hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. We identified an approximately 120-kDa calmodulin-binding protein that shares with hair-bundle myosin I the properties of being photolabeled by vanadate-trapped uridine nucleotides and immunoreactive with a monoclonal antibody raised against mammalian myosin I beta. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, we inhibited calmodulin action and measured the results with two distinct assays. Calmodulin antagonists increased photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolished adaptation to sustained mechanical stimuli. Our evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor.

Acyl glucuronide reactivity in perspective: biological consequences

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10% of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Nucleoside and oligonucleotide approaches towards potential HIV chemotherapies

The use of oligonucleotides directed against the mRNA of HIV promises site-specific inhibition of viral replication. In this work, the effect of aralkyl substituents on oligonucleotide duplex stability was studied using model oligonucleotide sequences in an attempt to improve targeting of oligonucleotides to viral mRNA. Arakyl-substituted oligonucleotides were made by solid phase synthesis using either the appropriate aralkyl-substituted phosphoramidite or by post-synthetic substitution of a pentafluorophenoxy substituent by N-methylphenethylamine. The presence of phenethyl or benzoyl substituents invariably resulted in thermodynamic destabilisation of all duplexes studied. The methods which were developed for the synthesis of nucleoside intermediates for oligonucleotide applications were also used to prepare a series of nucleoside analogues derived from uridine, 2'-deoxyuridine and AZT. Crystal structures of six compounds were successfully determined. Anti-HIV activity was observed for most compounds in the series although none were without cytotoxicity. The most active compound of the series was the ribose nucleoside; 1-β-D-erythro-pentofuranosyl-4-pentafluorophenoxy-pyrimidine-2(1H)-one 95, derived directly from uridine. The same series of compounds also displayed very modest anti-cancer activity. To enable synthesis of prooligonucleotides and analogues for possible antisense applications, the properties of a new Silyl-Linked Controlled Pore Glass solid support were investigated. Synthesis of the sequences d(Tp)7T, d(Tps)7T and the base-sensitive d(Tp)3(CBzp)2(Tp)2T was achieved using the silyl-linked solid support in a fluoride-induced cleavage/deprotection strategy.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

Developing of Germyldesulonylation and Thiodesulfonylation Reactions for the Synthesis of Novel Nucleoside Analogues. Efficient Synthesis of Novel (α-Fluoro)vinyl Sulfides

S-adenosyl-L-homocysteine (AdoHcy) hydrolase effects hydrolytic cleavage of AdoHcy to produce both adenosine and L-homocysteine and is a feedback inhibitor of S-adenosyl- L-methionine (SAM). Nucleoside analogues bearing an alkenyl or fluoroalkenyl chain between sulfur and C5' utilizing Negishi coupling reactions were synthesized. Palladium-catalyzed cross-coupling between the 5'-deoxy-5'-(iodomethylene) nucleosides and alkylzinc bromides gives analogues with the alkenyl unit. Palladium-catalyzed selective monoalkylation of 5'-(bromofluoromethylene)-5'-deoxy-adenosine with alkylzinc bromide afford adenosylhomocysteine analogues with a 6'-(fluoro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-L-homocysteine hydrolases. Stannydesulfonylation reaction is a critical step in the synthesis of E-fluorovinyl cytidine (Tezacitabine) a ribonucleoside reductase inhibitor with a potent anticancer activity. The synthesis involves the removal of the sulfonyl group by a radical-mediated stannyldesulfonylation reaction using tributyltin hydride. In order to eliminate the toxicity of tin, I developed a radical-mediated germyldesulonylation utilizing less toxic germane hydrides. Treatment of the protected (E)-5'-deoxy-5'-[(p-toluenesulfonyl)-methylene]uridine and adenosine derivatives with tributyl- or triphenylgermane hydride effected radical-mediated germyldesulfonylations to give 5'-(tributyl- or triphenylgermyl)methylene-5'-deoxynucleoside derivatives as single (E)-isomers. Analogous treatment of 2'-deoxy-2'-[(phenylsulfonyl)methylene]uridine with Ph3GeH afforded the corresponding vinyl triphenylgermane product. Stereoselective halodegermylation of the (E)-5'-(tributylgermyl)-methylene-5'-deoxy nucleosides with NIS or NBS provided the Wittig-type (E)-5'-deoxy-5'-(halomethylene) nucleosides quantitatively. Radical-mediated thiodesulfonylation of the readily available vinyl and (α-fluoro) vinyl sulfones with aryl thiols in organic or aqueous medium to provide a bench and environmentally friendly protocol to access (α-fluoro)vinyl sulfides were developed. Methylation of the vinyl or (α-fluoro)vinyl phenyl sulfide gave access to the corresponding vinyl or (α-fluoro)vinyl sulfonium salts. These sulfonium ions were tested as possible methyl group donors during reactions with thiols, phenols or amino groups which are commonly present in natural amino acids.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with β-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Safety and efficacy of ledipasvir-sofosbuvir in black patients with hepatitis C virus infection: A retrospective analysis of phase 3 data.

UNLABELLED: Black patients chronically infected with genotype 1 hepatitis C virus (HCV) have historically had lower rates of response to interferon-based treatment than patients of other races. In the phase 3 ION program, the single-tablet regimen of the NS5A inhibitor ledipasvir and NS5B nucleotide polymerase inhibitor sofosbuvir was shown to be safe and highly effective in the general population. The aim of this study was to evaluate the safety and efficacy of ledipasvir/sofosbuvir in black patients using data from the three open-label ION clinical trials, which evaluated the safety and efficacy of 8, 12, and 24 weeks of ledipasvir/sofosbuvir with or without ribavirin for the treatment of treatment-naïve and treatment-experienced patients with genotype 1 HCV, including those with compensated cirrhosis. The primary endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). For our analysis, rates of SVR12, treatment-emergent adverse events, and graded laboratory abnormalities were analyzed in black versus non-black patients. Of the 1949 patients evaluated, 308 (16%) were black. On average, black patients were older, had higher body mass index, were more likely to be IL28B non-CC, and had a lower serum alanine aminotransferase at baseline than non-black patients. Overall, 95% of black and 97% of non-black patients achieved SVR12. The rate of relapse was 3% in black patients as compared with 2% in non-black patients. The most common adverse events included fatigue, headache, nausea, and insomnia. The majority of adverse events occurred more frequently in the ribavirin-containing arms of the studies. No differences were observed in overall safety by race. CONCLUSION: A once-daily dosage of ledipasvir/sofosbuvir was similarly effective in black and non-black patients with genotype 1 HCV infection. The addition of ribavirin did not appear to increase SVR12 but was associated with higher rates of adverse events.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.