Chain Translocation Research Articles

Calculation of chemical potentials of chain molecules by the incremental gauge cell method

The gauge cell Monte Carlo method is extended to calculations of the incremental chemical potentials and free energies of linear chain molecules. The method was applied to chains of Lennard-Jones beads with stiff harmonic bonds up to 500 monomers in length. We show that the suggested method quantitatively reproduces the modified Widom particle insertion method of Kumar et al. [S. K. Kumar, I. Szleifer, and A. Z. Panagiotopoulos, Phys. Rev. Lett. 66(22), 2935 (1991)], and is by an order of magnitude more efficient for long chains in terms of the computational time required for the same accuracy of chemical potential calculations. The chain increment ansatz, which suggests that the incremental chemical potential is independent of the chain length, was tested at different temperatures. We confirmed that the ansatz holds only for coils above the θ temperature. Special attention is paid to the effects of the magnitude of adsorption potential and temperature on the behavior of single chains in confinements that are comparable in size with the free chain radius of gyration. At sufficiently low temperatures, the dependence of the incremental chemical potential on the chain length in wetting pores is superficially similar to a capillary condensation isotherm, reflecting monolayer formation following by pore volume filling, as the chain length increases. We find that the incremental gauge cell method is an accurate and efficient technique for calculations of the free energies of chain molecules in bulk systems and nanoconfinements alike. The suggested method may find practical applications, such as modeling polymer partitioning on porous substrates and dynamics of chain translocation into nanopores.

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃-S-ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

Primary Central Nervous System Burkitt Lymphoma With Non-Immunoglobulin Heavy Chain Translocation in Right Ventricle: Case Report

Primary central nervous system Burkitt lymphoma (PCNSBL) is rare. Few cases of primary central nervous system involvement with sporadic Burkitt lymphoma have been reported and its treatment is now controversial. Here, the authors report a case of a 14-year-old boy suffering from non-immunoglobulin heavy chain (IgH) translocation PCNSBL. To the authors’ knowledge, this is the second case report describing primary Burkitt lymphoma involving cerebral ventricles. After receiving combination treatment with surgery, stereotactic radiosurgery, and a chemotherapy regimen including high-dose methotrexate, the patient had a disease-free survival of 18 months.

English

  The activation and nuclear translocation of NF-kB and the expression of the pro-inflammatory cytokine genes by macrophages infected with the attenuated Brucella abortus RB51 and virulent 2308 strains were evaluated. pIkBα and NF-kB were determined by immunoblot, and cytokines IFN-g and IL12 mRNA were determined by reverse transcriptase polymerase chain reaction (qPCR) and translocation of NF-kB protein to the nucleus was determined by electrophoretic mobility shift assay (EMSA). We demonstrate that the attenuated B. abortus RB51 strain stimulates cells resulting in NF-kB activation and nuclear translocation, during experimental infection in macrophages J774A.1 which induced a pro-inflammatory response producing IL-6, 12 TNF-s INF-g and iNOS. The virulent strain B. abortus 2308 also stimulated the cells but induced a p50 homodimer of NF-kB which is inactive. The p50 homodimer of NF-kB binds to DNA, and thus blocked the activation of pro-inflammatory cytokines genes. Therefore, an evasion mechanism of the strain 2308 is to produce an inactive homodimer of NF-kB which does not give rise to pro-inflammatory response to eliminate the bacteria.   Key words: Brucella abortus, RB51, 2308, NF-kB, transduction signals.

Alpha‐lipoic acid does not alter stress protein response to acute exercise in diabetic brain

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.

Translocation of compact polymer chains through a nanopore

We perform dynamical Monte Carlo simulation to study the forced translocation of compact polymer chains in three-dimensional lattices. The chains are driven through a nanopore connecting two infinite channels by an external field. The scaling properties of average translocation time τ and translocation time distribution (TTD) are studied. The effects of contact energy (ɛC), electric field strength (E), and nanopore width (L) on the scaling exponent (α) of average translocation time τ∼Nα and the TTD are investigated. For the scaling behavior of τ∼Nα, we have found that there is no crossover behavior with weak field strength when the nanopore width is one lattice spacing, which is less than average bond length, while crossover behaviors are observed for larger nanopore widths. The scaling exponent α also depends on contact energy ɛC and electric field strength E. For the TTD, it shifts from the Gaussian to a right-skew distribution with the electric field E increasing for short chains; while for long chains, multi-peak distributions are observed. As a primary and simple model, compact polymer chains are extensively used to capture the structure and thermodynamic properties of proteins, therefore we can investigate the protein translocation by simulating compact chain translocation, and this study will be useful for exploring the complex translocation behaviors of proteins.

On the Roles of Anionic Lipids in Protein Localization and Permeability of Membranes

Anionic lipids, such as phosphatidylglycerol and phosphatidylserine, play important structural and functional roles in cell membranes. In particular, they appear to provide strong interactions with positively charged protein side chains to promote membrane localization of lipid binding domains and antimicrobial peptides, as well as to modulate the function of many membrane proteins. All-atom molecular dynamics simulations were used to explore the strength of these interactions and the impact they have on the ability of charged protein residues to penetrate into membranes. Using an analog of arginine and bilayers of pure phosphatidylcholine or mixtures with phosphatidylglycerol, we have computed the thermodynamics of charged side chain translocation, as well as the binding affinity of each lipid within the membrane. We found that arginine deforms the bilayer in a similar fashion, regardless of composition, and that the free energy profile for translocation is relatively unaffected by anionic lipids: the “neutralization” of the protein side chain does not reduce the large ∼20 kcal/mol barrier significantly. We decomposed these free energies to explain why anionic lipids do not play a significant role, with implications for the actions of many charged peptides and ion permeability. We also find that arginine binding to phosphatidylglycerol is more favorable by only ∼1 kcal/mol, suggesting that lipid binding domains and antimicrobial peptides likely require many charged side chains acting together to promote membrane localization.

Bimodal modulation of the botulinum neurotoxin protein-conducting channel

Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.

Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Many proteins are translocated across and integrated into the endoplasmic reticulum membrane. The type I signal anchor sequence mediates the translocation of its preceding region through the endoplasmic reticulum membrane, but the source of the motive force has been unclear. Here, we characterized the motive force for N-terminal domain translocation using two probes. First, an Ig-like domain of the muscle protein titin (I27 domain) or its mutants were fused to the N termini, and translocation was examined in a cell-free translation system supplemented with rough microsomal membrane. The N-terminal translocation efficiencies correlated with the mechanical instabilities of the I27 mutants. When the I27 domain was separated from the signal anchor sequence by inserting a spacer, even the most unstable mutant stalled on the cytoplasmic side, whereas its downstream portion spanned the membrane. Proline insertion into the signal anchor sequence also caused a large translocation defect. Second, a streptavidin-binding peptide tag was fused to the N terminus. Titration of streptavidin in the translation system allowed us to estimate the translocation motive force operative on the tag. The motive force was decreased by the proline insertion into the signal anchor sequence as well as by separation from the signal anchor sequence. When the streptavidin-binding peptide tag was separated from the signal anchor, the proline insertion did not induce further deficits in the motive force for the tag. On the basis of the findings obtained by using these two independent techniques, we conclude that the signal sequence itself provides the motive force for N-terminal domain translocation within a limited upstream region.

OH-2, a Hyperdiploid Myeloma Cell Line without An IGH Translocation, Has a Complex Translocation Juxtaposing MYC near MAFB and the IGK Locus

Despite heterogeneity of phenotypes, multiple myeloma (MM) can be classified into two major groups: hyperdiploid (HRD) tumors with 48–74 chromosomes, which typically have extra copies of at least four of the eight odd chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; and non-hyperdiploid (NHRD) tumors, which usually have immunoglobulin heavy chain (IGH) translocations and <46 or >74 chromosomes. Human myeloma cell lines (HMCL) with a typical HRD genotype are lacking. The OH-2 HMCL was derived from extramedullary myeloma, and retains the same HRD phenotype as the primary tumor, with extra copies of chromosomes 3, 7, 15, 19, and 21, as demonstrated by array comparative genomic hybridization. This provides a unique example of an HMCL and the corresponding primary tumor that share the same HRD phenotype. Also Spectral Karyotyping showed the same HRD phenotype. By fluorescence in situ hybridization it was shown that OH-2 does not have an IGH or Ig lambda light chain translocation. Instead, both the HMCL and the primary tumor have complex translocations involving chromosomes 2, 8, and 20. The translocation juxtaposes MAFB near MYC and the Ig kappa (IGK) enhancer. Both MYC and MAFB are highly expressed, as shown by microarray analysis, and are thought to be dysregulated by the IGK enhancer. Both OH-2 HMCL and the primary tumor cells express high levels of Cyclin D2, an expected consequence of the enhanced MAFB level. OH-2 has a phenotype similar to the 20% of HRD MM tumors that express increased levels of Cyclin D2 but not Cyclin D1 and could be a good in vitro model for studies of this subgroup of MM. OH-2 is dependent on medium containing human serum and growth-promoting cytokines like IL-6 or IL-21, reflecting the critical role of the microenvironment for growth of MM cells.

Scaling Law of Poly(ethylene oxide) Chain Permeation through a Nanoporous Wall

This paper presents a study of the permeation of poly(ethylene oxide) (PEO) chains through the nanoporous wall of hollow polymeric capsules prepared by self-assembly of polyelectrolytes. We employ the method of pulsed field gradient (PFG) NMR diffusion to distinguish chains in different sites, i.e., in the capsule interior and free chains in the dispersion, by their respective diffusion coefficient. From a variation of the observation time, the time scale of the molecular exchange between both sites and thus the permeation rate constant is extracted from a two-site exchange model. Permeation rate constants show two different regimes with a different dependence on chain length. This suggests a transition between two different mechanisms of permeation as the molecular weight is increased. In either regime, the permeation time can be described by a scaling law tau approximately N (b) , with b = (4)/ 3 for short chains and b = (1)/ 3 for long chains. We discuss these exponents, which clearly differ from the theoretical predictions for chain translocation.

Aggressive CD5-positive B-cell lymphoma after remission of CD5-negative follicular lymphoma with distinct immunoglobulin heavy chain rearrangement and translocation

We report a unique, aggressive B-cell lymphoma that developed after the long-term remission of follicular lymphoma (FL). FL cells were negative for CD5, whereas aggressive lymphoma cells were positive for CD5. In FL, one immunoglobulin heavy chain gene (IGH) allele underwent V/D/J recombination and another t(14;18)(q32;q21). In aggressive lymphoma, one IGH allele underwent D/J recombination and another translocation, but not t(14;18)(q32;q21). An aggressive lymphoma-specific D/J sequence was detected in FL tissue. Our results indicated that the two tumors arose from distinct B cells and that they existed concurrently in the same lymph node.

TransCGH: Rapid Identification and High-Resolution Mapping of Balanced IgH Translocations in Archival DNA Using Custom Oligonucleotide Arrays.

Array based comparative genomic hybridization (CGH) has revolutionized the study of chromosomal imbalances but generally is incapable of detecting balanced genomic rearrangements like reciprocal translocations, which play central roles in the pathogenesis and diagnosis of lymphomas, leukemias and other tumors. The precise identification of immunoglobulin heavy chain (IgH) translocation partners, for example, is essential for the classification of B cell lymphomas and for predicting prognosis in plasma cell neoplasms like multiple myeloma. Using IgH translocations as a model for balanced genomic rearrangements, we have developed a simple modification of array CGH that we call translocation-CGH (transCGH) and that enables the rapid identification of IgH translocation partners and precise mapping of translocation-associated breakpoints to unprecedented resolution. To render IgH translocations detectable on CGH arrays, genomic DNA from test and reference samples is modified prior to array hybridization in an enzymatic linear amplification reaction that employs a single IgH joining (JH) or switch (Sμ/Sα/Sε) region primer, resulting in specific amplification of any fusion partner sequences that may be inserted (via translocation or other rearrangement) downstream of the IgH primer. Using a single tiling-density oligonucleotide array representing such common IgH partner loci as MYC, BCL2 and CCND1 (cyclin D1), transCGH successfully identified and mapped to ∼100bp resolution an assortment of known IgH fusion breakpoints in various cell lines and primary lymphomas, including JH-CCND1 breakpoints in MO2058 and Granta 519 cell lines (mantle cell lymphoma), a cytogenetically cryptic Sα-CCND1 fusion in U266 (myeloma), JH-MYC and Sμ-MYC breakpoints in MC116 and Raji (Burkitt lymphoma), and JH-BCL2 breakpoints in DHL16 (large cell lymphoma; minor cluster region) and in an archival case of follicular lymphoma (major breakpoint region). We then used transCGH to analyze 4 archival cases of mantle cell lymphoma and one t(11;14)-positive case of B cell prolymphocytic leukemia, all of which lacked PCR-detectable translocation breakpoints at the CCND1 major translocation cluster (MTC). Five novel CCND1 translocation breakpoints were identified and mapped to ∼100bp resolution, allowing the rapid design of patient-specific PCR primers for amplification, sequencing, and confirmation of the predicted breakpoints. One breakpoint mapped to within 500bp of the MTC, whereas the other 4 were scattered across a ∼150kb region flanking the MTC. To our knowledge, this represents the largest series of non-MTC mantle cell lymphoma breakpoint sequences reported to date. It also illustrates how transCGH can facilitate the rapid cloning of previously unidentified IgH translocation breakpoints dispersed over very large genomic regions. Because transCGH requires only genomic DNA and can simultaneously detect both balanced IgH translocations and genomic imbalances at ultra-high resolution on the same array, it may become a useful alternative to molecular cytogenetic methods (e.g. FISH) for clinical testing of B cell and plasma cell neoplasms. transCGH also will facilitate the development of highly sensitive breakpoint-specific PCR assays for detecting minimal residual disease. Finally, because the primer used in the linear amplification reaction is fully customizable, transCGH can readily be adapted to identify and map other balanced translocations (or more complex genomic fusions) that involve non-IgH loci, provided that one of the fusion partners is known.

Translocation of polymers with folded configurations across nanopores

The transport of polymers with folded configurations across membrane pores is investigated theoretically by analyzing simple discrete stochastic models. The translocation dynamics is viewed as a sequence of two events: motion of the folded segment through the channel followed by the linear part of the polymer. The transition rates vary for the folded and linear segments because of different interactions between the polymer molecule and the pore. It is shown that the translocation time depends nonmonotonously on the length of the folded segment for short polymers and weak external fields, while it becomes monotonous for long molecules and large fields. Also, there is a critical interaction between the polymers and the pore that separates two dynamic regimes. For stronger interactions, the folded polymer moves slower, while for weaker interactions, the linear chain translocation is the fastest. In addition, our calculations show that the folding does not change the translocation scaling properties of the polymer. These phenomena can be explained by the interplay between translocation distances and transition rates for the folded and linear segments of the polymer. Our theoretical results are applied for analysis of experimental translocations through solid-state nanopores.

Translocation of polymer chains through interacting nanopores

The effect of the interaction between nanopore and chain segment on the translocation of polymer chains through an interacting nanopore from a confined environment (cis side, high concentration of chain) to a spacious environment (trans side, zero concentration) was studied by using dynamic Monte Carlo simulations. Results showed that a moderate attractive pore–polymer interaction accelerates the translocation of chain. The optimal interaction at which translocation is the fastest increases with the concentration of chain on the cis side. The dependence of microscopic behaviors of chain translocation on the interaction was investigated.

Interferon-γ enhances superoxide production in human mesangial cells via the JAK–STAT pathway

Immune reactive cytokines, such as interferon (IFN)-gamma, have multiple effects in glomerulonephritis. Superoxide anions (O(2)(-)), which are associated with the progression of glomerulonephritis, are mainly generated by nicotinamide adenine dinucleotide phosphate (reduced form) NAD(P)H oxidases. We determined the effects of IFN-gamma on O(2)(-) production, phosphorylation of signal transducer and activator of transcription (STAT)-1alpha, and the mRNA and protein expressions of p22phox and Nox1, components of NAD(P)H oxidases, in human mesangial cells (HMCs). Significant increases in O(2)(-) production with IFN-gamma were completely abolished by the flavin-containing enzyme inhibitor, diphenyleneiodonium (10 micromol/l), and the Janus-activated kinase (JAK)2 inhibitor, AG490 (100 micromol/l). Phosphorylated STAT-1alpha was detected after 5 min of IFN-gamma stimulation using Western blot analysis, and binding to the gamma-activating site was observed from 30 min to 4 h, thereafter by electrophoretic mobility shift assay (EMSA). Super-shift analysis in EMSA revealed that the main transcription factor was STAT-1alpha. IFN-gamma significantly increased the expression of p22phox mRNA and protein, although expression was inhibited by AG490. These data suggest that IFN-gamma stimulates O(2)(-) production in HMCs via the JAK-STAT pathway and NAD(P)H oxidase.

Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHASE327Q and seHASE327K were expressed at low levels, whereas seHASE327D and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHASE327K,K48E protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (approximately 0.6 MDa) was made by seHASE327K,K48E and seHASK48E. The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

Molecular heterogeneity of sporadic adult Burkitt-type leukemia/lymphoma as revealed by PCR and cytogenetics: correlation with morphology, immunology and clinical features

Chromosomal translocations involving the MYC oncogene are a hallmark of Burkitt lymphoma but they are only found in a varying frequency in mature Burkitt-type acute lymphoblastic leukemia (B-ALL). We have investigated samples of 56 sporadic Burkitt leukemia/lymphoma patients for the translocations t(8;14)(q24;q32), t(2;8)(p11;q24) and t(8;22)(q24;q11). Long PCR was used for detecting the immunoglobulin heavy chain (IgH) translocation and cytogenetics and/or fluorescence in situ hybridization for detecting the 'variant' MYC translocations. A total of 29 samples (51.8%) were t(8;14)-positive by long PCR. Approximately one-third had a chromosomal breakpoint in the IgH joining region while the others had breakpoints in the IgH switch regions. Among them were two cases with a previously unreported MYC translocation into the IgE switch region. Long PCR was more reliable compared to conventional cytogenetics for detecting the t(8;14). Epstein-Barr virus was detected in high copy number in two (3.6%) t(8;14)-positive cases by real-time quantitative PCR. Human herpesvirus 8 was not detected in any case by nested PCR. A typical L3 or L3-compatible cytomorphology was highly predictive (>80%) but not specific of a MYC translocation. A total of 34 patients were treated according to the GMALL B-ALL therapy protocols and there was no significant difference in overall survival between patients with or without t(8;14).

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.