Light-inducible flux control of triosephosphate isomerase on glycolysis in Escherichia coli.

An engineering tool for controlling flux distribution on metabolic pathways to an appropriate state is highly desirable in bioproduction. An optogenetic switch, which regulates gene expression by light illumination is an attractive on/off switchable system, and is a promising way for flux control with an external stimulus. We demonstrated a light-inducible flux control between glycolysis and the methylglyoxal (MGO) pathway in Escherichia coli using a CcaS/CcaR system. CcaR is phosphorylated by green light and is dephosphorylated by red light. Phosphorylated CcaR induces gene expression under the cpcG2 promoter. The tpiA gene was expressed under the cpcG2 promoter in a genomic tpiA deletion strain. The strain was then cultured with glucose minimum medium under green or red light. We found that tpiA messenger RNA level under green light was four times higher than that under red light. The repression of tpiA expression led to a decrease in glycolytic flux, resulting in slower growth under red light (0.25 hr -1 ) when compared to green light (0.37 hr -1 ). The maximum extracellular MGO concentration under red light (0.2 mM) was higher than that under green light (0.05 mM). These phenotypes confirm that the MGO pathway flux was enhanced under red light.

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli.

Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Šresolution, which is a significant improvement over the previous structure.

Human glyceraldehyde-3-phosphate dehydrogenase in man-rodent somatic cell hybrids.

The human enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms heteropolymers with the rodent enzyme in man-rodent somatic cell hybrids. A gene specifying GAPDH is syntenic with the genes specifying the glycolytic enzymes triosephosphate isomerase (TPI) and lactate dehydrogenase B (LDH-B). The synteny of GAPDH, TPI, and LDH-B is the first evidence for the syntenic association of human genes that specify enzymes of the Embden-Meyerhof glycolytic pathway.

At least one intron is required for the nonsense-mediated decay of triosephosphate isomerase mRNA: a possible link between nuclear splicing and cytoplasmic translation.

Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3'-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3'-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3'-most intron from pre-mRNA "marks" the 3'-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the "mark" mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5' untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.

Three new crystal structures of point mutation variants of monoTIM: conformational flexibility of loop-1, loop-4 and loop-8.

BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop. The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity. RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor. These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8. In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure. CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized. In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis.

Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses.

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.

Cytoplasmic mRNA for human triosephosphate isomerase is immune to nonsense-mediated decay despite forming polysomes.

Nonsense codons between position 14 within the first exon and position 193 within the penultimate exon of the human gene for triosephosphate isomerase reduce mRNA abundance to 25% of normal. The reduction in abundance is due to the decay of newly synthesized mRNA that copurifies with nuclei. TPI mRNA that copurifies with cytoplasm is immune to decay. We show here that immunity is not due to the failure of nonsense-containing mRNA to form polysomes. This finding indicates that cytoplasmic mRNA, in contrast to nucleus-associated mRNA, may have lost one or more factors that are required for nonsense-mediated decay or gained one or more factors that confer immunity to nonsense-mediated decay.

Cloning of the triosephosphate isomerase gene of Plasmodium falciparum and expression in Escherichia coli.

A major supply of energy in the rapidly multiplying intraerythrocytic Plasmodium falciparum is from the glycolytic pathway. We have isolated the cDNA and genomic clones of the glycolytic enzyme, triosephosphate isomerase (TPI) by polymerase chain reaction (PCR). Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences derived from TPIs of other organisms, were used to prime PCR on P. falciparum DNA. The P. falciparum TPI gene is interrupted by a single intron which divides the coding region into two exons. The coding region encodes a protein of 248 amino acids which is of the same size as TPIs from other organisms and shares 42-45% homology with other known eukaryotic TPIs. On comparison with human TPI the catalytic domain was found to be highly conserved, while significant variations occurred at the other regions in the protein sequence. The P. falciparum TPI gene was cloned into the expression vector pTrc99A and hyperexpressed as an unfused protein in Escherichia coli. The 28-kDa protein was shown to be catalytically active.

The use of antibodies for characterization and quantification of a recombinant protein.

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica.

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a ß-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. ß-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.

Unravelling the bull fertility proteome.

In the last few decades a negative association between the level of milk production and fertility has been observed. Currently, the most utilized method of measuring male fertility employed by the livestock industry is related to the Non-Return Rate (NRR). Through differential proteome analysis, this study evaluated changes in the expression of the protein profile of spermatozoa collected from 16 bulls with different levels of field fertility expressed as an estimated relative conception rate (ERCR). The main aim is to identify putative protein markers to be used as putative indices of fertility. Two dimensional electrophoresis coupled with mass spectrometry analysis was used for protein separation and identification. To improve differential proteome analysis among experimental groups, a part of shotgun MS analysis was also performed. Three protein spots showed a differential expression pattern among all ERCR classes. Alpha enolase was significantly down-regulated in the ERCR- group, while two other proteins, isocitrate dehydrogenase and triosephosphate isomerase, were up-regulated in ERCR- in comparison to ERCR+. Alpha-enolase and isocitrate dehydrogenase subunit alpha (IDH-alpha) have been described in the literature for having a potential role in bull fertility. The possibility of determining protein biomarkers for fertility is more useful and less expensive than ERCR for acquiring rapid estimation of fertility because it does not require the use of field insemination trials. Shotgun MS analysis conducted on the same samples revealed 7 proteins down-regulated in the ERCR- group and 1 protein up-regulated. Among these proteins, calmodulin, ATP synthase mitochondrial subunits alpha and delta, malate dehydrogenase and sperm equatorial segment protein 1 were shown to be linked with sperm fertility.

G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT.

In G-CSF-mobilized hematopoietic SCT (HSCT), natural killer (NK) cells have a critical role in GVHD and GVL effects. However, regulation of NK cell response to G-CSF remains unclear. This study assayed G-CSF effects in both HSCT donors and NK-92MI cells. The donors who received G-CSF had significantly decreased NK cell cytotoxicity. Levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated (p)-Akt, but not mammalian target of rapamycin (mTOR), were downregulated in NK cells from G-CSF-injected donors. G-CSF also decreased cytotoxicity without affecting viability and NF-κB of NK-92MI cells. PI3K and p-ERK expression were also decreased in G-CSF-treated NK-92MI cells, and their inhibitors, wortmannin and PD98059, respectively, both enhanced the downregulation of cytotoxicity. These effects were accompanied by decreased expression of a cytotoxicity-related gene, triosephosphate isomerase (TPI). Wortmannin, but not PD98059, enhanced the downregulation of TPI in G-CSF-treated NK-92MI cells, indicating a correlation between PI3K and TPI. We conclude that G-CSF-impaired NK cell cytotoxicity may accompany PI3K/Akt signaling. The effect is transient and NK cells may recover after G-CSF clearance, suggesting that G-CSF-mobilized HSCT may benefit both acute GVHD prevention and late-phase GVL promotion in HSCT recipients.

Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice.

As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min (-1) mg (-1) which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.

Hominoid triosephosphate isomerase: characterization of the major cell proliferation specific isozyme.

Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [35S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains a blocked NH2-terminus and length heterogeneity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.

Intron position affects expression from the tpi promoter in rice.

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the first tpi intron, and the gene encoding bacterial beta-glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the rice tpi gene enhanced expression of the GUS gene from the tpi promoter when it was placed 5' of the GUS gene. When the tpi intron was placed in the 3'-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.

The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules.

The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.

The Lactococcus lactis triosephosphate isomerase gene, tpi, is monocistronic.

Triosephosphate isomerase (EC 5.3.1.1) from Lactococcus lactis was purified to electrophoretic homogeneity. Approximately 3 mg purified enzyme (specific activity 3300 U mg-1) was obtained from 70 g (wet wt) cells. In solution, triosephosphate isomerase (pI 4.0-4.4) was observed to exist as a homodimer (M(r) 57,000) of noncovalently linked subunits. The sequence of the first 37 amino acid residues from the NH2-terminus were determined by step-wise Edman degradation. This sequence, and that of a region conserved in all known bacterial triosephosphate isomerases, was used to design oligonucleotide primers for the synthesis of a lactococcal tpi probe by PCR. The probe was used to isolate a molecular clone of tpi from a lambda GEM11 library of L. lactis LM0230 DNA. The nucleotide sequence of tpi predicted a protein of 252 amino acids with the same NH2-terminal sequence as that determined for the purified enzyme and a subunit M(r) of 26,802 after removal of the NH2-terminal methionine. Escherichia coli cells harbouring a plasmid containing tpi had 15-fold higher triosephosphate isomerase activity than isogenic plasmid-free cells, confirming the identity of the cloned gene. Northern analysis of L. lactis LM0230 RNA showed that a 900 base transcript hybridized with tpi. The 5' end of the transcript was determined by primer extension analysis to be a G located 65 bp upstream from the tpi start codon. These transcript analyses indicated that in L. lactis, tpi is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to tpi did not encode another Embden-Meyerhoff-Parnas pathway enzyme. The location of tpi on the L. lactis DL11 chromosome map was determined to be between map coordinates 1.818 and 1.978.

Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein.

Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting the alpha/beta-barrel fold. In a previous work, a monomeric mutant of hTIM was engineered in which Met14 and Arg98, two interface residues, were changed to glutamine. Analysis of equilibrium denaturation of this monomeric mutant, named M14Q/R98Q, revealed that its conformational stability, 2.5kcal/mol, is low as compared to the stability of dimeric hTIM (19.3 kcal/mol). The fact that this value is also lower than the conformational stabilities usually found for monomeric proteins suggests that the hTIM monomers are thermodynamically unstable. In the present work, we attempted to stabilize the M14Q/R98Q mutant by introducing stabilizing mutations in alpha-helices of the protein. Five mutations were proposed, designed to increase alpha-helix propensity by introducing alanines at solvent-exposed sites (Q179A, K193A), to introduce favorable interactions with helix dipoles (Q179D, S105D), or to reduce the conformational entropy of unfolding by introducing proline residues at the "N-cap" position of alpha-helices (A215P). Three replacements (Q179D, K193A, and A215P) were found to increase the stability of the native dimeric hTIM and the monomeric M14Q/R98Q. These results suggest that the monomeric hTIM mutant can be stabilized to a considerable extent by following well-established rules for protein stabilization. A comparison of the stabilizing effect performed by the mutations on the dimeric hTIM and the monomeric M14Q/R98Q allowed us to reinforce a model of equilibrium denaturation proposed for both proteins.

Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA.

The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to 20-30% of normal by frameshift and nonsense mutations that prematurely terminate translation within the first three-quarters of the reading frame. The decrease has been shown to be attributable to a reduced level of TPI mRNA that copurifies with nuclei. Given that the translational reading frame of an mRNA is assessed in the cytoplasm during protein synthesis, cytoplasmic and nuclear RNA processes may be linked. Alternatively, a nuclear mechanism may exist whereby in-frame nonsense codons can be identified. To differentiate between these two possibilities, two distinct modulators of protein synthesis have been tested for the ability to influence the nonsense-codon-mediated reduction in the mRNA level. (i) A suppressor tRNA, which acts in trans to suppress an amber nonsense codon within TPI mRNA, and (ii) a hairpin structure in the 5' untranslated region of TPI mRNA, which acts exclusively in cis to inhibit initiation of TPI mRNA translation, were found, individually, and to a greater extent, together, to abrogate the decrease in mRNA. These results show that tRNA and ribosomes coordinately mediate the effect of a nonsense codon on the level of newly synthesized TPI mRNA. We suggest that the premature termination of TPI mRNA translation in the cytoplasm can reduce the level of TPI mRNA that fractionates with nuclei.