MST1/2 in inflammation and immunity.

The mammalian Sterile 20-like kinase 1/2 (MST1/2) belongs to the serine/threonine (GC) protein kinase superfamily. Collective studies confirm the vital role MST1/2 in inflammation and immunity. MST1/2 is closely related to the progress of inflammation. Generally, MST1/2 aggravates the inflammatory injury through MST1-JNK, MST1-mROS, MST1-Foxo3, and NF-κB pathways, as well as several regulatory factors such as tumor necrosis factor-α (TNF-α), mitochondrial extension factor 1 (MIEF1), and lipopolysaccharide (LPS). Moreover, MST1/2 is also involved in the regulation of immunity to balance immune activation and tolerance by regulating MST1/2-Rac, MST1-Akt1/c-myc, MST1-Foxos, MST1-STAT, Btk pathways, and lymphocyte function-related antigen 1 (LFA-1), which subsequently prevents immunodeficiency syndrome and autoimmune diseases. This article reviews the effects of MST1/2 on inflammation and immunity.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

eEF-2K knockdown synergizes with STS treatment to inhibit cell proliferation, migration, and invasion via the TG2/ERK pathway in A549 cells.

Emerging research has suggested the anticancer potential of tanshinone IIA, the bioactive ingredient isolated from the traditional Chinese herb Salvia miltiorrhiza. However, the molecular mechanism of sodium tanshinone IIA sulfonate (STS) antilung cancer effect is not very clear. In this study, our purpose is to investigate the roles of STS and elongation factor-2 kinase (eEF-2K) in regulating the proliferation, migration, and invasion of A549 cells and explore the implicated pathways. We found that STS suppressed A549 cell survival and proliferation in a time- and xdose-dependent manner. Knockdown of eEF-2K and treatment with STS synergistically exerted antiproliferative, -migratory, and -invasive effects on A549 cells. These effects were caused by attenuation of the extracellular signal-regulated kinase (ERK) pathway via inhibition of tissue transglutaminase (TG2). In summary, the inhibition of eEF-2K synergizes with STS treatment, exerting anticancer effects on lung adenocarcinoma cells through the TG2/ERK signaling pathway, which provides a potential therapeutic target for treating lung adenocarcinoma.

Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli.

The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37°C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496mg/L of NusA-fused C5aRA could be produced.

Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis.

Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.

Glial restricted precursors protect against chronic glutamate neurotoxicity of motor neurons in vitro.

We have examined the expression of glutamate transporters in primary and immortalized glial precursors (GRIPs). We subsequently transduced these cells with the GLT1 glutamate transporter and examined the ability of these cells to protect motor neurons in an organotypic spinal cord culture. We show that glial restricted precursors and GRIP-derived astrocytes predominantly express glutamate transporters GLAST and GLT1. Oligodendrocyte differentiation of GRIPs results in downregulation of all glutamate transporter subtypes. Having identified these precursor cells as potential vectors for delivering glutamate transporters to regions of interest, we engineered a line of GRIPS that overexpress the glutamate transporter GLT1. These cells (G3 cells) have a nearly fourfold increase in glutamate transporter expression and at least a twofold increase in the V(max) for glutamate transport. To assess whether G3 seeding can protect motor neurons from chronic glutamate neurotoxicity, G3s were seeded onto rat organotypic spinal cord cultures. These cultures have previously been used extensively to understand pathways involved in chronic glutamate neurotoxicity of motor neurons. After G3 seeding, cells integrated into the culture slice and resulted in levels of glutamate transport sufficient to enhance total glutamate uptake. To test whether neuroprotection was related to glutamate transporter overexpression, we isolated GRIPS from the GLT1 null mouse to serve as controls. The seeding of G3s resulted in a reduction of motor neuron cell death. Hence, we believe that these cells may potentially play a role in cell-based neuroprotection from glutamate excitotoxicity.

A carboxy-terminal domain of ELL is required and sufficient for immortalization of myeloid progenitors by MLL-ELL.

The t(11;19)(q23;p13.1) chromosomal translocation in acute myeloid leukemias fuses the gene encoding transcriptional elongation factor ELL to the MLL gene with consequent expression of an MLL-ELL chimeric protein. To identify potential mechanisms of leukemogenesis by MLL-ELL, its transcriptional and oncogenic properties were investigated. Fusion with MLL preserves the transcriptional elongation activity of ELL but relocalizes it from a diffuse nuclear distribution to the nuclear bodies characteristic of MLL. Using a serial replating assay, it was demonstrated that the MLL-ELL chimeric protein is capable of immortalizing clonogenic myeloid progenitors in vitro after its retroviral transduction into primary murine hematopoietic cells. However, a structure-function analysis indicates that the elongation domain is not essential for myeloid transformation because mutants lacking elongation activity retain a potent ability to immortalize myeloid progenitors. Rather, the highly conserved carboxyl terminal R4 domain is both a necessary and a sufficient contribution from ELL for the immortalizing activity associated with MLL-ELL. The R4 domain demonstrates potent transcriptional activation properties and is required for transactivation of a HoxA7 promoter by MLL-ELL in a transient transcriptional assay. These data indicate that neoplastic transformation by the MLL-ELL fusion protein is likely to result from aberrant transcriptional activation of MLL target genes. Thus, in spite of the extensive diversity of MLL fusion partners, these data, in conjunction with previous studies of MLL-ENL, suggest that conversion of MLL to a constitutive transcriptional activator may be a general model for its oncogenic conversion in myeloid leukemias. (Blood. 2000;96:3887-3893)

An ATPase center of rat liver 30S-5SRNP particles.

Treatment of 30S-5SRNP with 1 M Cs(2)SO(4) at 2 degrees C overnight followed by sucrose density-gradient centrifugation yielded particles smaller than 30S-5SRNP, designated as CsS-particles. CsCl density-gradient centrifugation of CsS-particles showed the homogeneity of the particles containing about half the amount of proteins in 30S-5SRNP particles. The particles contained 18SrRNA, 5SRNP and about half the number of proteins in 30S-5SRNP. The ATPase activity of freshly prepared CsS-particles was about half the original 30S-5SRNP level although it was unstable even at 2 degrees C. Poly(U) slightly enhanced the activity, and phe-tRNA(phe) stimulated it concentration-dependently. EF-1a alone enhanced it, and in combination with poly(U) and phe-tRNA(phe) stimulated it markedly. EF-2 alone markedly increased it. The activity with the full components for elongation described above became very high, being comparable to that of the original 30S-5SRNP and twice that of 40S subunits. A two-dimensional electrophoretogram of the protein in CsS-particles revealed 9 small subunit protein species, in addition to L5, which included proteins interacting with mRNA and two elongation factors. Taken together with the results of our preceding study indicating the participation of ATPase of 80S ribosomes in peptide elongation, the present results indicate CsS-particles may be a part of the ATPase centre of 80S ribosomes.

Effects of the active aldehyde group generated by RNA N-glycosidase in the sarcin/ricin domain of rat 28S ribosomal RNA on peptide elongation.

Effects of the active aldehyde group of ribose C1' at position 4324 of rat 28S rRNA, in the inactivated ribosome generated by RNA N-glycosidases (trichosanthin, A-chain of cinnamomin and ricin), on peptide elongation have been studied. The aldehyde group inhibits the activities of eEF1A-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent GTPase, but increases eEF2-dependent activity. At a high concentration of RNA N-glycosidase, the generated aldehyde group also inhibits aminoacyl-tRNA binding to the inactivated ribosome in the absence of elongation factor and translocation activity. When the aldehyde group is reduced into a hydroxyl group by sodium borohydride or blocked with an amino acid through nucleophilic addition, the activities of eEF1A-dependent aminoacyl-tRNA binding and eEF1A-dependent GTPase of the inactivated ribosome are partially restored, but the altered activities of eEF2-dependent GTPase, translocation and aminoacyl-tRNA binding in the absence of elongation factor are not normalized. Thus, reduction or blockage of the aldehyde group with sodium borohydride or amino acids might change the conformation of the S/R domain in rat 28S ribosomal RNA to meet the requirement for eEF1A-dependent reactions, but not eEF2-involved reactions.

Elongation factor-1alpha stabilizes microtubules in a calcium/calmodulin-dependent manner.

Elongation factor-1alpha (EF-1alpha), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of EF-1alpha (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1alpha modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1alpha, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1alpha significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1alpha, occur. In addition, calcium/calmodulin (Ca2+/CaM), which regulates the ability of EF-1alpha to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1alpha on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1alpha was never observed. These data support the hypothesis that EF-1alpha modulates the dynamic behavior of microtubules assembled in vitro in a Ca2+/CaM-dependent manner.

ATPase associated with ribosomal 30S-5SRNP particles and 40S subunits of rat liver.

The ATPase activity of rat liver 30S-5SRNP particles prepared by EDTA treatment of 80S ribosomes, and that of 40S subunits were investigated in correlation with polypeptide elongation. The ATPase activity of 30S-5SRNP particles was higher than that of 40S subunits. Poly(U) and TMV RNA stimulated the ATPase activity of 30S-5SRNP particles more markedly than that of 40S subunits. These two kinds of particles also showed intrinsic GTPase. Poly(U) enhanced the GTPase activity of 30S-5SRNP particles but not that of 40S subunits. An elongation factor (EF-1alpha, EF-2, or EF-1alphabetagamma) alone or in combination with poly(U) and/or other elongation factors stimulated the ATPase activities of both particles. The extent of stimulation of the ATPase activity by a combination of these components was usually somewhat higher than or similar to the sum of those with the individual components. The extents of stimulation by these components were higher in the case of 30S-5SRNP particles than that of 40S subunits, indicating the importance of the 5SRNP moiety in the former particles. The intactness of 18SrRNA was required for promotion of the ATPase activity of 30S-5SRNP particles by Phe(+), (-)tRNA(Phe). The ATPase activities of the two kinds of particles by themselves or those observed with the combinations of the components mentioned above were inhibited by several kinds of translation inhibitors. The degrees of inhibition were generally higher for 30S-5SRNP particles. The ATPase activity of 40S subunits was enhanced by spermidine, suggesting the importance of the conformational change induced by it. These results imply the participation of the intrinsic ATPase of 30S-5SRNP particles and 40S subunits in polypeptide elongation, and the important role of the 5SRNP moiety of 30S-5SRNP particles in the ATPase activity.

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends.

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.

Tet(M)-promoted release of tetracycline from ribosomes is GTP dependent.

Tet(M) protein, which displays homology to elongation factor G (EF-G), interacts with the protein biosynthetic machinery to render this process resistant to tetracycline in vivo and in vitro. To clarify the basis of the resistance mechanism, the effects of Tet(M) on several reactions which occur during protein synthesis were examined. The mechanism of action of Tet(M) has been clarified by two observations. The protein relieves tetracycline inhibition of factor-dependent tRNA binding and dramatically reduces the affinity of ribosomes for tetracycline when GTP is present. This reduction in drug affinity appears to be due to a large increase in the rate of tetracycline dissociation. Addition of Tet(M) to ribosome-tetracycline complexes results in displacement of bound drug. And, while Tet(M) and EF-G GTPase activities are tetracycline resistant, the two proteins differ in their sensitivities to fusidic acid, with the latter activity inhibited by the drug. Furthermore, while Tet(M) protects translation from tetracycline inhibition in a defined system, it is unable to substitute for either EF-G or elongation factor Tu.

F-actin bundling activity of Tetrahymena elongation factor 1 alpha is regulated by Ca2+/calmodulin.

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome. Previously, Tetrahymena 14-nm filament-associated protein was identified as EF-1 alpha [Kurasawa et al. (1992) Exp. Cell Res. 203, 251-258]. This and several other studies suggest that EF-1 alpha functions not only in translation but also in regulation of some part of the cytoskeleton. Tetrahymena EF-1 alpha bound to F-actin and induced bundling of F-actin. We investigated the effects of GTP/GDP and Ca2+/calmodulin on F-actin bundling activity of EF-1alpha. The presence of GTP, GDP, or guanylyl-imidodiphosphate (GMP-PNP) slightly decreased the amount of EF-1 alpha which bound to F-actin, but each had virtually no effect on the F-actin bundling activity. The formation of F-actin bundles by EF-1 alpha was Ca(2+)-insensitive. In the absence of Ca2+, calmodulin did not bind to EF-1 alpha and F-actin. On the other hand, in the presence of Ca2+, calmodulin directly bound to EF-1 alpha but did not have any serious influence on EF-1 alpha/F-actin binding. Under the conditions, electron microscopy demonstrated that Ca2+/calmodulin completely inhibited the F-actin bundling by EF-1 alpha. These results indicate that CA2+/calmodulin regulates the F-actin bundling activity of EF-1 alpha without inhibition of the binding between Ef-1 alpha and F-actin.

Drosophila RNA polymerase II mutants that affect transcription elongation.

We have examined the properties of two Drosophila RNA polymerase II mutants, C4 and S1, during elongation, pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid substitution in the largest and second largest subunits, respectively. Compared with wild type, C4 had a lower elongation rate and was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and NH4+ whereas the S1 mutant was less responsive to both. Differences between the two mutants also appeared during DmS-II-mediated transcript cleavage and pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at generating the final pattern of shortened transcripts. S1 and wild type were equal in the rate of extended DmS-II mediated transcript cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in elongation competent mode and that the C4 mutation may affect the movement of the polymerase.

Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase.

Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis.

Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.

A factor protecting mammalian [75Se]SeCys-tRNA is different from EF-1 alpha.

In Escherichia coli, an elongation factor (EF-Tu-like) specific to SeCys-tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys-tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 x g from bovine liver. The proteins were separated by Sephacryl S-300 chromatography, and the SePF and EF-1 alpha activities were found in the same fraction, indicating that SePF and EF-1 alpha have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM-Sephadex C-25 columns. The SePF activity was eluted after the peak of EF-1 alpha activity. This result indicated that this SePF activity was not dependent on EF-1 alpha. In addition to performing these two chromatographies, we investigated pure EF-1 alpha from Bombyx mori but could not detect any SePF activity in B. mori EF-1 alpha. Thus we showed that the SePF activity in bovine liver differs from that of EF-1 alpha in eukaryotes. Therefore the factor protecting [75Se]SeCys-tRNA in bovine liver is not EF-1 alpha and must be a SELB-like factor.

Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site.

mRNA analogues containing 4-thiouridine residues at selected sites were used to extend our analysis of photo-induced cross-links between mRNA and 16S RNA to cover the entire downstream range between positions +1 and +16 on the mRNA (position +1 is the 5'-base of the P-site codon). No tRNA-dependent cross-links were observed from positions +1, +2, +3 or +5. Position +4 on the mRNA was cross-linked in a tRNA-dependent manner to 16S RNA at a site between nucleotides ca 1402-1415 (most probably to the modified residue 1402), and this was absolutely specific for the +4 position. Similarly, the previously observed cross-link to nucleotide 1052 was absolutely specific for the +6 position. The previously observed cross-links from +7 to nucleotide 1395 and from +11 to 532 were however seen to a lesser extent with certain types of mRNA sequence from neighbouring positions (+6 to +10, and +10 to +13, respectively); no tRNA-dependent cross-links to other sites on 16S RNA were found from these positions, and no cross-linking was seen from positions +14 to +16. In each case the effect of a second cognate tRNA (at the ribosomal A-site) on the level of cross-linking was studied, and the specificity of each cross-link was confirmed by translocation experiments with elongation factor G, using appropriate mRNA analogues.

Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components.

Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.