Ribosomal Salt Wash Research Articles

Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE

Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro, and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.

A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes.

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem-loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA(Phe) or its anticodon stem- loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.

Yeast Ribosomal Protein L12 Is a Substrate of Protein-arginine Methyltransferase 2

Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogeneity. The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2. Using S-adenosyl-l-methionine as co-substrate, RMT2 methylated a protein in the ribosome salt wash fraction. The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease. Amino acid analysis affirmed that the labeling products were delta-N-monomethylarginines. The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry. Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg(67) on ribosomal protein L12 as the methyl acceptor. L12 was isolated from wild type yeast cells that have been grown in the presence of S-adenosyl-l-[methyl-(3)H]methionine and subjected to amino acid analysis. The results indicate that L12 contains delta-N-monomethylarginines.

Mutational analysis of the different bulge regions of hepatitis C virus domain II and their influence on internal ribosome entry site translational ability.

The hepatitis C virus (HCV) 5'-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.

Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs

Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al., 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MA P2- R NA t rans - a cting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted α-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the α-subunit of the Ca 2+/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.

Ribosome-mediated Folding of Partially Unfolded Ricin A-chain

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.

Purification and Characterization of β-Adrenergic Receptor mRNA-binding Proteins

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.

Initiation of protein synthesis with fluorophore-Met-tRNA f and the involvement of IF-2

The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA f during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA f with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA f determined that all were capable of binding IF-2 at 4 mM Mg 2+ and 37 °C. Filter binding assays conducted in the absence of magnesium ions on fMet-tRNA f, eosin-Met-tRNA f, and cascade yellow-Met-tRNA f confirmed the previously reported value for the dissociation constant of fMet-tRNA f of about 1 μM and placed the binding constants for the two fluorophore derivatives about three-fold higher. Binding of the fluorophore-Met-tRNA f species to salt-washed ribosomes showed a more significant decrease compared to fMet-tRNA f. Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF-2 was observed in each case. All ribosome-bound cascade yellow-Met-tRNA f and eosin-Met-tRNA f were as puromycin-reactive as fMet-tRNA f. Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides.

Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Nuclear factor-90 of activated T-cells: A double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR.

NFAT transcription factors play a central role in initiating T-cell activation through the induction of immediate-early T-cell specific genes including interleukin-2 (IL-2). NFAT transcription factors bind to a sequence in the IL-2 enhancer known as the antigen receptor response element 2 (ARRE-2). Multiple proteins exhibiting ARRE-2 binding activity have been isolated, including a heterodimer from stimulated T-cell nuclear extracts consisting of Mr = 90 000 (NF90) and Mr = 45 000 (NF45) subunits. The subunits of this heterodimer have been cloned, and NF90 was found to encode a protein containing two domains that are predicted to form motifs capable of binding to double-stranded RNA. Using in vitro translated polypeptides, we have demonstrated that NF90 specifically binds to double-stranded RNA. Furthermore, NF90 was phosphorylated in a double-stranded RNA-dependent manner likely by the interferon-induced, double-stranded RNA-dependent protein kinase, PKR. The NF90 protein was found to be expressed not only in T-cells, but also in nonimmune HeLa cells. In HeLa cells, the protein was almost exclusively localized to the ribosome salt wash fraction of cell lysates.

Complex Formation by All Five Homologues of Mammalian Translation Initiation Factor 3 Subunits from Yeast Saccharomyces cerevisiae

The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.

Accumulation of ppGpp in Streptococcus pyogenes and Streptococcus rattus following amino acid starvation.

We have re-examined the stringent response of Streptococcus rattus and Streptococcus pyogenes, two organisms that had originally been reported not to accumulate ppGpp following amino acid deprivation. We conclude that ppGpp does accumulate when S. rattus and S. pyogenes are deprived of isoleucine by mupirocin addition. The kinetics of ppGpp accumulation was faster in S. pyogenes compared with S. rattus. Cell fractionation and analysis of in vitro ppGpp synthesis showed that in S. pyogenes most activity was associated with the S-100 ribosomal pellet, whereas in S. rattus the S-100 soluble fraction contained greater activity. The addition of 20% methanol or salt-washed ribosomes to the assay mixture did not stimulate the in vitro (p)ppGpp synthesis activity of fractions isolated from S. rattus or S. pyogenes. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that neither organism cross-reacted under conditions that detected RelA in E. coli CF1648. However, cross-reaction with anti-RelSeq antibody was observed in S. pyogenes but not S. rattus, suggesting that ppGpp synthesis is carried out by a putative SpoT protein in S. pyogenes and by a functionally unknown protein in S. rattus.

Accumulation of ppGpp in Streptococcus pyogenes and Streptococcus rattus following amino acid starvation

We have re-examined the stringent response of Streptococcus rattus and Streptococcus pyogenes, two organisms that had originally been reported not to accumulate ppGpp following amino acid deprivation. We conclude that ppGpp does accumulate when S. rattus and S. pyogenes are deprived of isoleucine by mupirocin addition. The kinetics of ppGpp accumulation was faster in S. pyogenes compared with S. rattus. Cell fractionation and analysis of in vitro ppGpp synthesis showed that in S. pyogenes most activity was associated with the S-100 ribosomal pellet, whereas in S. rattus the S-100 soluble fraction contained greater activity. The addition of 20% methanol or salt-washed ribosomes to the assay mixture did not stimulate the in vitro (p)ppGpp synthesis activity of fractions isolated from S. rattus or S. pyogenes. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that neither organism cross-reacted under conditions that detected RelA in E. coli CF1648. However, cross-reaction with anti-Rel Seq antibody was observed in S. pyogenes but not S. rattus, suggesting that ppGpp synthesis is carried out by a putative SpoT protein in S. pyogenes and by a functionally unknown protein in S. rattus.

2] Preparation and application of chaperone-deficient Escherichia coli cell-free translation systems

Publisher Summary The chapter discusses the role of chaperones in folding of nascent proteins, either as they are synthesized on ribosomes or immediately after they are terminated and released. Five bacterial chaperones—DnaJ, DnaK, GrpE, GroEL, and GroES—can promote folding and release of full-length rhodanese that was not properly folded and released from Escherichia coli ( E. coli ) ribosomes after its synthesis. The chapter discusses whether this is the major pathway by which nascent proteins are folded into their native conformation. E. coli salt-washed ribosomes, their 50S subunit, or even the 23S rRNA in its native state can efficiently refold denatured rhodanese independent of Adenosine-5'-triphosphate (ATP). The results relate the ability of ribosomes to carry out refolding to the peptidyltransferase center and their ability to form peptide bonds. To test the hypothesis that ribosomes themselves can promote folding of nascent proteins without the assistance of molecular chaperones, in vitro systems for protein synthesis that are deficient in molecular chaperones are developed. The aim is to develop a system that totally lacks these proteins. The description of the experimental procedures addresses the following three aspects: (1) preparation of the cell-free systems, (2) methods to quantitate the chaperones that remain in these systems, and (3) in vitro synthesis of test proteins and their analysis, including their enzymatic activity.

Developmental regulation of a plant encoded inhibitor of eukaryotic initiation factor 2 alpha phosphorylation.

An inhibitor of eIF-2a phosphorylation was identified in various plant species. The plant protein (termed PKI) specifically cross-reacts with monoclonal antiserum that recognizes the glycosylated, active form of a M(r) 87 kD protein analog (p67) from reticulocytes. Northern blot analysis using a probe to the reticulocyte inhibitor cDNA further supports the presence of analogous transcripts in plant tissue. PKI specifically inhibits the phosphorylation of the plant encoded eIF-2 alpha kinase (pPKR) as well as plant and human eIF-2 alpha phosphorylation. The interaction between PKI and pPKR is indicated by their copurification on dsRNA agarose, despite evidence showing that PKI does not bind dsRNA. Further, wheat PKI inhibits human PKR phosphorylation but activity is recovered by immuno-depletion of PKI from wheat germ fractions. PKI is temporally regulated during plant growth and development. It is maximally present in extracts from dormant seeds, however, it is not detectable soon after leaf emergence at approximately 48 h post-imbibition. PKI levels are again detectable at the mid-milk stage in seed development. Protein levels of pPKR in ribosomal salt wash and cytosolic extracts from healthy plant tissue remain essentially constant throughout the life cycle. In contrast, pPKR activity levels based upon autophosphorylation vary significantly and are inversely correlated with PKI protein levels. Phosphorylation of eIF-2 alpha is a classical mechanism for the downregulation of protein synthesis suggesting that inhibition of pPKR activity by PKI may contribute to the dramatic and rapid increase in protein synthesis observed during seed germination.

Determination of Eukaryotic Peptidyltransferase Activity by Pseudo-First-Order Kinetic Analysis

We have developed anin vitrosystem for the determination of peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA–poly(U)–80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate (k3) constant is identical to the catalytic rate constant (kcat) of peptidyltransferase. Thisk3of peptidyltransferase is equal to 2.9 min−1at 37°C. Moreover, the ratiok3/Ks, which is an accurate measure of peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic peptidyltransferase.

The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is prevented by eIF4E binding protein (PHAS-I; 4E-BP1) in the reticulocyte lysate.

A common feature of viral infection is the subversion of the host cell machinery towards the preferential translation of viral products. In some instances, this is partly mediated by the expression of virally encoded proteases which lead to the cleavage of initiation factor eIF4G. The foot-and-mouth disease virus encodes two forms of a cysteine proteinase (L protease) which bisects the eIF4G polypeptide into an N-terminal fragment containing the eIF4E binding site, and a C-terminal fragment which contains binding sites for eIF4A and eIF3 and which associates with the 40S ribosomal subunit. Previously, we have demonstrated that the cleavage of eIF4G by L protease stimulates the translation of uncapped transcripts encoding cellular proteins and supports internal initiation driven by picornavirus internal ribosome entry segment (IRES) elements. Use of reticulocyte lysates manipulated to deplete them of eIF4E and the N-terminal fragment suggests that the C-terminal fragment of eIF4G is responsible for these effects, and we have now confirmed this by purifying the C-terminal fragment and analysing its effects directly in the absence of L protease. Interestingly, we find that pre-incubation of reticulocyte lysates or ribosomal salt wash fractions with the specific eIF4E binding protein, PHAS-I (eIF4E-BP1), blocks the proteolytic cleavage of eIF4G by L protease. This effect can be reversed by addition of recombinant eIF4E. These data are consistent with a model whereby the L protease cleavage site in eIF4G is inaccessible until a change in conformation is induced by the binding of eIF4E. This may have implications for a role for eIF4E binding in triggering changes that expose other domains in the eIF4G molecule during initiation of translation.

Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.