Helical Elements Research Articles

Flux enhancement during Dean vortex tubular membrane nanofiltration: 13. Effects of concentration and solute type

Controlled centrifugal instabilities (called Dean vortices) resulting from sufficient flow in composite polyamide–poly(ether sulfone) helical membrane tubes have been used to reduce concentration polarization during nanofiltration. These vortices enhance back-migration through convective flow away from the membrane–solution interface and increased shear at the membrane–solution interface and allow for increased membrane permeation rates. As a result, solute concentrations at the membrane–solution interface and resulting osmotic-driven back flow are reduced. The performance of two sets of modules (designated Set II and Set III), each set containing a prototype vortex generating helical tubular nanofiltration (NF) element and a conventional linear element was evaluated. Nanofiltration of aqueous solutions of inorganic salts (including KCl, K 2SO 4 and K 3PO 4) and amino acids of similar molecular weight (including glutamic acid, glutamine and lysine) was performed with Set II. These experiments, designed to evaluate the effects of solute type, were conducted at the same energy consumption and transmembrane pressures. Both membrane swelling and charge effects were evident as a function of varying the pH during membrane filtration of both inorganic salts and amino acids. Both flux and rejection were higher for the helical module than the linear module during amino acid nanofiltration. A new modified phenomenological model was shown to be effective for predictive purposes for cases of responsive concentration polarization. Its applicability is validated by performing nanofiltration of aqueous MgSO 4 solutions with a new set of modules designated as Set III. Modules of Set III contained dissimilar helical and linear elements. The model was then tested against the results obtained previously.

The hydrogen bonded polymer structures of [{Mn(2-mbiH)2·TMEDA}·A–A]∞ [2-mbiH2 = 2-mercaptobenzimidazole; A–A = TMEDA (Me2NCH2CH2NMe2) or DABCO (N{CH2CH2}3N)

The reaction of [MnCp2] with 2-mbiH2 (1:2 equiv.) in the presence of TMEDA gives [{Mn(2-mbiH)2·TMEDA}·TMEDA]∞1. A similar reaction employing TMEDA and DABCO (1:1 equiv.) yields [{Mn(2-mbiH)2·TMEDA}·DABCO]∞2. In the crystal, both complexes adopt similar polymeric structures in which the bifunctional [Mn(2-mbiH)2·TMEDA] units are associated by hydrogen bonds to lattice-bound TMEDA or DABCO ligands. The structural motif adopted by both complexes can be regarded as resultingfrom a fusion of separate helical elements.

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41.

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577-599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization.

Evolutionary variation in bacterial RNase P RNAs.

Sequences encoding RNase P RNAs from representatives of the last remaining classical phyla of Bacteria have been determined, completing a general phylogenetic survey of RNase P RNA sequence and structure. This broad sampling of RNase P RNAs allows some refinement of the secondary structure, and reveals patterns in the evolutionary variation of sequences and secondary structures. Although the sequences range from 100 to <25% identical to one another, and although only 40 of the nucleotides are invariant, there is considerable conservation of the underlying core of the RNA sequence. RNase P RNAs, like group I intron RNAs but unlike ribosomal RNAs, transfer RNAs or other highly conserved RNAs, are quite variable in secondary structure outside of this conserved structural core. Conservative regions of the RNA evolve by substitution of apparently interchangeable alternative structures, rather than the insertion and deletion of helical elements that occurs in the more variable regions of the RNA. In a remarkable case of convergent molecular evolution, most of the unusual structural elements of type B RNase P RNAs of the low G+C Gram-positive Bacteria have evolved independently in Thermomicrobium roseum , a member of the green non-sulfur Bacteria.

Studies on the large subunit rRNA genes and their flanking regions of leuconostocs

The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris were amplified by polymerase chain reactions and sequenced. The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp. dextranicum were also sequenced. The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions. A single tRNA(Ala) gene without the 3'-terminal CCA sequence was found in spacer-1 regions. Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs. For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing. Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers. Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents. Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures. Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs. We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA-spacer-1-tRNA(Ala)-23S rRNA-spacer-2-5S rRNA-3'.

Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase.

The pyridoxal-P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal-P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo- and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form most of the conserved fold of the pyridoxal-P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo- and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.

Solution structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85α subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85α N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 Cα atoms of 1.09 and 0.89 Å, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85α recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (βB5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (βD5) in C-SH2 to Ile, the corresponding residue type in the p85α N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85α N-SH2. This pattern of p85α phosphopeptide binding specificity is interpreted in terms of a model of the p85α/PDGF-receptor interaction.

EXPLORING CLATHRATE FORMATION WITH AN OPTICALLY RESOLVED HOST. X-RAY CRYSTAL STRUCTURE OF THE INCLUSION COMPOUND BETWEEN (11S, 12S)-(-)-9,10-DIHYDRO-9 ,10-ETHANOANTHRACENE-11,12-DICARBOXYLIC ACID AND N-HEXANE (2 : 1)

The crystal structure of the inclusion compound formed between (11S,12S)-(-)-9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboxylic acid, (1), and n-hexane (2:1) has been studied by X-ray diffraction. It crystallizes in the tetragonal space group P41 and represents a less common type of inclusion compound, which has helical and chiral structural elements. Helical chains, formed by hydrogen-bonded host molecules, wind around the 21 screw axes and encircle the guest molecules. Crystal data: a=b=17.478(1); c=12.021(1)A, Z=4 host–guest 2:1 units, R=0.043, Rw=0.061 for 2225 observations with I > 3σ (I). The general shape and conformational flexibility of 1 with respect to the requirements of inclusion formation and crystal packing are discussed.

Studies on the large subunit rRNA genes and their flanking regions of leuconostocs

The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris were amplified by polymerase chain reactions and sequenced. The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp. dextranicum were also sequenced. The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions. A single tRNAAla gene without the 3'-terminal CCA sequence was found in spacer-1 regions. Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs. For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing. Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers. Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents. Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures. Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs. We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA - spacer-1 - tRNAAla - 23S rRNA - spacer-2 - 5S rRNA-3'.Key words: leuconostocs, rrn operons, rDNA, 23S, spacer regions, polymerase chain reaction.

PH responsive microdomain formation in a de novo polypeptide.

Recombinant DNA technology has been employed to produce a polypeptide capable of forming pH responsive hydrophobic microdomains. The design of this peptide is based upon an idealized conceptual model in which electrostatic, hydrophobic, and hydration forces are responsible for the association of amphipathic alpha-helical elements. Reduction in solution pH is responsible for reducing electrostatic repulsions between similarly charged residues, promoting the hydrophobic collapse of helical elements. A polymerizable synthetic element (dn31) has been synthesized and inserted into an appropriate expression vector. A clone containing a single copy of the dn31 gene (designated dn3lx1) was isolated and the corresponding gene product DN3Lx1 isolated. The physical properties of DN3Lx1 were examined in solution by gel filtration chromatography. CD, and fluorescence probe analysis. It was determined that DN3Lx1 self-associates in solution with the degree of aggregation dependent on pH and ionic strength. An initial objective of this work was to examine domain organization in higher molecular weight species containing ten or more repetitive sequences. However, attempts to express multiple repeats of DN3Lxn from concatemers were unsuccessful.

Structure of the C-terminal Fragment 300-320 of the Rat Angiotensin II AT1A Receptor and Its Relevance with Respect to G-Protein Coupling

Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha-helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.

Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg2+ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements. Increased concentration of Mg2+ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na+ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg2+.

Sequence Effects on RNA Bulge-Induced Helix Bending and a Conserved Five-Nucleotide Bulge from the Group I Introns

Bulge loops introduce bends in RNA double helices. Thus, a role for bulge loops in the tertiary folding of RNA is to orient helical elements. The location, size, and sequence of a five-nucleotide bulge are conserved in many of the self-splicing group I introns. We have used gel electrophoretic analysis of helix bending to test the hypothesis that this bulge loop is conserved to control the angle between the flanking helices. Interruption of an RNA duplex by the five-nucleotide bulge of the group I intron from Tetrahymena thermophila results in an electrophoretically retarded species, indicative of bending by the bulge. However, mutation of conserved bases in the bulge has a small effect on the retardation, suggesting that the average induced bend angle is not strongly dependent on the conserved sequence. Electrophoretic analysis of a mixture of bulged duplexes containing all five-nucleotide bulges reveals that most five-nucleotide bulge sequences induce bends that are similar to the bend induced by the conserved bulge. We have calibrated relative electrophoretic mobilities with bends of known magnitude, and characterized the distribution of bulge sequences among bend angles. Though the entire range of bend angles induced by different five-nucleotide bulges is from approximately 45 degrees to 75 degrees, most ( > 85%) five-nucleotide bulge loops induce bends between 65 degrees and 75 degrees. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65 degrees. They are generally, though not universally, pyrimidine-rich.

Getting closer to an understanding of the three-dimensional structure of ribosomal RNA.

Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.

Applications of high-T/sub c/ superconductors in sub-Gigahertz r.f. cavity resonators

The size of cavity resonators below 1 GHz may be reduced by using dielectric or helical loading. With normal conductors, such electrically small resonators suffer problems of losses and inadequate Q-factor. However, by using superconductor in the portions with the highest current density, very good performance may be obtained with very electrically-small cavities. A cylindrical dielectric resonator was used to load a corrugated cavity, having YBaCuO thick-film end plates, and operated at VHF. The results showed a significant improvement over corrugated cavities with copper end plates. Investigations have also been undertaken with high-T/sub c/ superconductor helical loading. The size of the cavities was electrically very small (about 0.02 of free space wavelength), and results with both bulk and thick-film helical loading elements were obtained. The results showed a significant improvement over a similar copper helically-loaded cavity resonator.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Mechanical behaviour of the metallic elements of submarine cables as a function of cable loading

Submarine power cables are widely used for local and national distribution and it is of interest to know their mechanical limitations particularly with regard to the loads experienced during the installation process where static or low-cycle fatigue failures are possible. A detailed understanding of the global and internal mechanics of such cables is therefore of interest in order to achieve the required performance at reasonable weight. This paper addresses the relationship between applied loads and the responses of the metallic elements within typical subsea configurations. The existing theory on mechanics of cables of this type is extended to take into account strain distributions within helical elements and to model the interactions between loading modes as well as including nonlinear effects associated with plasticity in the metallic elements and compliance of the polymeric material within the composite construction. The results of a number of material tests are incorporated into the new analysis and the predictions of the model are also compared with measurements on the conductors of cables under conditions of axial and bending loading.

A Temperature-dependent Conformational Rearrangement in the Ribosomal Protein S4˙16 S rRNA Complex

Ribosomal protein S4 protects a characteristic set of bases in 16 S rRNA from attack by chemical probes. Use of hydroxyl radical as a probe of the RNA backbone shows that ribose residues in these same regions are also protected by S4, confirming the localization of its interactions with 16 S rRNA to the junction of five helical elements in the proximal region of the 5' domain. At 0 degrees C, the nucleotides protected by S4 from base-specific probes are confined almost exclusively to the two compound helices formed by residues 404-499. After subsequent heating of the complex briefly at 30 or 42 degrees C, nucleotides in the three adjacent helices are additionally protected, resulting in a pattern of protection that is identical to that which is observed when S4 is incubated with 16 S rRNA under in vitro reconstitution conditions. Preincubation of the protein or the RNA (or both) separately at elevated temperature does not substitute for heating the S4.RNA complex. The regions in the RNA affected by the heat step are known to interact with proteins S12 and S16, both of which depend upon S4 for their binding to the RNA. Thus, the finding that S4 recruits additional sites of interaction in the RNA following its initial binding suggests a possible mechanism to insure the sequential addition of proteins during ribosomal assembly.

A helical element in the C-terminal domain of the N. plumbaginifolia F1 beta presequence is important for recognition by the mitochondrial processing peptidase.

The mitochondrial processing peptidase (MPP) in lower eucaryots and mammals is a matrix enzyme, whereas MPP in plants constitutes an integral part of the bc1 complex of the respiratory chain. The isolated spinach leaf bc1 complex catalyzes cleavage of the precursor of Nicotiana plumbaginifolia F1 beta subunit of the ATP synthase, resulting in a production of mature protein and a presequence that consists of 54 amino acids. A synthetic peptide derived from the C-terminal part of the presequence, containing 17 amino acids with a helical structural element, p-F1 beta(38-54), was an efficient inhibitor of the processing, whereas a peptide derived from the N-terminal part of the presequence, p-F1 beta(1-18), was much less effective. ATIII, a helical peptide derived from antithrombin III, was not recognized by MPP. Synthetic peptides corresponding to 4, 6, and 11 amino acids of the C terminus of the presequence, p-F1 beta(51-54), p-F1 beta(49-54), and p-F1 beta(44-54) were almost completely inert. Competition studies show that MPP recognizes the C-terminal domain of the presequence rather than the N-terminal domain. Furthermore, the alpha-helical element of the C-terminal domain is shown to be required for the recognition event.

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.