Polyphenylalanine Synthesis Research Articles

The natural bicyclic hexapeptide RA-VII is a novel inhibitor of the eukaryotic translocase eEF2

A cyclic hexapeptide, RA-VII isolated from the Rubiaceae family of plants, has high cytotoxic activity. Although RA-VII has been shown to inhibit protein synthesis in eukaryotic cells, the molecular mode of its action is not clear. Here we investigate the mechanism of the RAVII action on the translation apparatus. Biochemical functional assays showed that RA-VII inhibits poly(U)-dependent polyphenylalanine synthesis in the presence of animal elongation factors eEF1A and eEF2. Furthermore, RAVII prevented eEF2/ribosome-dependent GTPase activity, but not eEF-1A/ribosome-dependent activity. A filter binding assay demonstrated that RA-VII markedly enhances the binding affinity of eEF2 for GTP, but not for GDP, and prevents exchange of GTP in the eEF2-GTP complex, even after addition of a large excess of GTP/GDP. Limited proteolysis experiments indicated that RA-VII prevents the digestion of eEF2 in the presence of either GTP or GMPPCP, but not with GDP. Further footprint analysis and a translocation assay showed that the eEF2•GMPPNP•RA-VII complex binds to the conserved rRNA regions at the factor-binding center of the ribosome and retains the ability to translocate the A site-bound tRNA to the P-site. These results suggest that RA-VII tightly stabilizes the GTP•eEF2 complex structure, which is able to bind to the ribosomal functional site, but seems to suppress normal turnover of eEF2 after translocation. The properties of RA-VII make it a novel ligand for probing the action of eEF2 in the process of translocation on the ribosome.

The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation.

Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by 15N–1H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9–32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Å from the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.

Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.

Structural and Mutagenesis Studies Evince the Role of the Extended Protuberant Domain of Ribosomal Protein uL10 in Protein Translation.

The lateral stalk of ribosomes constitutes the GTPase-associated center and is responsible for recruiting translation factors to the ribosomes. The eukaryotic stalk contains a P-complex, in which one molecule of uL10 (formerly known as P0) protein binds two copies of P1/P2 heterodimers. Unlike bacterial uL10, eukaryotic uL10 has an extended protuberant (uL10ext) domain inserted into the N-terminal RNA-binding domain. Here, we determined the solution structure of the extended protuberant domain of Bombyx mori uL10 by nuclear magnetic resonance spectroscopy. Comparison of the structures of the B. mori uL10ext domain with eRF1-bound and eEF2-bound ribosomes revealed significant structural rearrangement in a "hinge" region surrounding Phe183, a residue conserved in eukaryotic but not in archaeal uL10. 15N relaxation analyses showed that residues in the hinge region have significantly large values of transverse relaxation rates. To test the role of the conserved phenylalanine residue, we created a yeast mutant strain expressing an F181A variant of uL10. An in vitro translation assay showed that the alanine substitution increased the level of polyphenylalanine synthesis by ∼33%. Taken together, our results suggest that the hinge motion of the uL10ext domain facilitates the binding of different translation factors to the GTPase-associated center during protein synthesis.

Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors.

Reconstitution of ribosomes in vitro from individual ribosomal proteins provides a powerful tool for understanding the ribosome assembly process including the sequential incorporation of ribosomal proteins. However, conventional assembly methods require high-salt conditions for efficient ribosome assembly. In this study, we reconstituted 30S ribosomal subunits from individually purified ribosomal proteins in the presence of ribosome biogenesis factors. In this system, two GTPases (Era and YjeQ) facilitated assembly of a 30S subunit exhibiting poly(U)-directed polyphenylalanine synthesis and native protein synthesis under physiological conditions. This in vitro system permits a study of the assembly process and function of ribosome biogenesis factors, and it will facilitate the generation of ribosomes from DNA without using cells.

Internal initiation of polyuridylic acid translation in bacterial cell-free system

The task of the present work was to answer the question: is the free 5'-end needed for effective translation of a model polyribonucleotide template - polyuridylic acid - in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5'-end and the polyuridylic acid with blocked 5'-end were compared in the bacterial cell-free translation system. To block the 5'-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5'-end and uridylic oligoribonucleotide sequence at its 3'-end, schematically described as FAM(dC)10(rU)50, was covalently attached (ligated) to the 5'-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5'-blocked template and on the polyuridylic acid with free 5'-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5'-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.

Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins

In animal ribosomes, two stalk proteins P1 and P2 form a heterodimer, and the two dimers, with the anchor protein P0, constitute a pentameric complex crucial for recruitment of translational GTPase factors to the ribosome. To investigate the functional contribution of each copy of the stalk proteins, we constructed P0 mutants, in which one of the two C-terminal helices, namely helix I (N-terminal side) or helix II (C-terminal side) were unable to bind the P1–P2 dimer. We also constructed ‘one-C-terminal domain (CTD) stalk dimers’, P1–P2ΔC and P1ΔC–P2, composed of intact P1/P2 monomer and a CTD-truncated partner. Through combinations of P0 and P1–P2 variants, various complexes were reconstituted and their function tested in eEF-2-dependent GTPase and eEF-1α/eEF-2-dependent polyphenylalanine synthesis assays in vitro. Double/single-CTD dimers bound to helix I showed higher activity than that bound to helix II. Despite low polypeptide synthetic activity by a single one-CTD dimer, its binding to both helices considerably increased activity, suggesting that two stalk dimers cooperate, particularly in polypeptide synthesis. This promotion of activity by two stalk dimers was lost upon mutation of the conserved YPT sequence connecting the two helices of P0, suggesting a role for this sequence in cooperativity of two stalk dimers.

PpGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.

Differential effects of paromomycin on ribosomes of Leishmania mexicana and mammalian cells.

Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis

The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.

The N‐terminal regions of eukaryotic acidic phosphoproteins P1 and P2 are crucial for heterodimerization and assembly into the ribosomal GTPase‐associated center

Acidic phosphoproteins P1 and P2 form a heterodimer and play a crucial role in assembly of the GTPase-associated center in eukaryotic ribosomes and in ribosomal interaction with translation factors. We investigated the structural elements within P1 and P2 essential for their dimerization and for ribosomal function. Truncation of the N-terminal 10 amino acids in either P1 or P2 and swapping of the N-terminal 10 amino acid sequences between these two proteins disrupted their dimerization, binding to P0 and P0 binding to rRNA. In contrast, truncation of the C-terminal halves of P1 and P2 as well as swapping of these parts between them gave no significant effects. The protein dimers containing the C-terminal truncation mutants or swapped variants were assembled with P0 onto Escherichia coli 50 S subunits deficient in the homologous protein L10 and L7/L12 and gave reduced ribosomal activity in terms of eukaryotic elongation factor dependent GTPase activity and polyphenylalanine synthesis. The results indicate that the N-terminal 10 amino acid sequences of both P1 and P2 are crucial for P1-P2 heterodimerization and for their functional assembly with P0 into the GTPase-associated center, whereas the C-terminal halves of P1 and P2 are not essential for the assembly.

Binding Mode of the First Aminoacyl-tRNA in Translation Initiation Mediated by Plautia stali Intestine Virus Internal Ribosome Entry Site

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Inorganic polyphosphate interacts with ribosomes and promotes translation fidelity in vitro and in vivo

Inorganic polyphosphate is a biological macromolecule consisting of multiple phosphates linked by high-energy bonds. Polyphosphate occurs in cells from all domains of life, and is known to play roles in a diverse collection of cellular functions. Here we examine the relationship between polyphosphate and protein synthesis in Escherichia coli. We report that polyphosphate associates with E. coli ribosomes in vitro. Characterization of this interaction reveals that both long-chain and short-chain polyphosphates interact with the ribosome. Intact 70S ribosomes, as well as 50S and 30S subunits, display a specific interaction with polyphosphate that is mediated primarily by contacts with ribosomal proteins. Additionally, we examined functional consequences of a ppk mutation, which severely reduces levels of intracellular polyphosphate. Extracts from ppk mutants contain lower levels of polysomes than wild-type cells, suggesting a defect in mRNA utilization or the mRNA-ribosome interaction. Ribosomes from wild-type and ppk mutant cells were isolated, and their activities were compared using a polyU RNA in vitro translation assay. While rates of polyphenylalanine synthesis are similar, use of ribosomes from ppk cells results in a misincorporation rate about five times higher compared with the rate observed when ribosomes from wild-type cells are used. Mistranslation rates in vivo were measured directly, and ppk mutants displayed higher readthrough frequencies for two different stop codons. Taken together, these results indicate that polyphosphate plays an important role in maintaining optimal translation efficiency in vivo and in vitro.

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.

Actin—an inhibitor of eukaryotic elongation factor activities

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger. Some component(s) in the postribosomal supernatant (S-130) fraction and also DNase I prevent actin-promoted inhibition of polyPhe synthesis.

Protein synthesis by single ribosomes.

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

KMP016 Molecular biological analysis of Interferon resistance in hepatitis C virus genotype 1b

An elongation factor with activity corresponding to that of mammalian EF-2 (avian EF-2) has been purified 500-fold from hen oviduct to apparent homogeneity using a 6-step procedure. The homogeneity of the preparation has been determined by the following criteria: (1) a single band on three polyacrylamide disc gel electrophoretic systems (anodic, cathodic, and sodium dodecyl sulfate); (2) velocity sedimentation ultracentrifugation; and (3) amino terminal analysis. These studies show that purified avian EF-2 is a single polypeptide. The purified protein has been found to have activity in the following reactions: (1) synthesis of polyphenylalanine from [14C]Phe-tRNA in the presence of rabbit reticulocyte EF-1; (2) formation and release of the dipeptide[14C]phenylalanylpuromycin also in the presence of EF-1; (3) the ribosome-dependent hydrolysis of [γ-32P]GTP; and (4) the diphtheria toxin-dependent [3H]ADP-ribose incorporation from [3H]NAD+. The activity of the factor in polypeptide synthesis is inhibited by fusidic acid and ADP-ribosylation. Avian EF-2 was inactive in stimulating polyphenylalanine synthesis from [14C]Phe-tRNA in a protein synthesis system derived from Escherichia coli.The molecular weight was determined to be 80 000 by band sedimentation ultracentrifugation, 96 000 by Sephadex G-200 gel filtration chromatography and 93 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Stokes radius was estimated to be 37.5 Å. The calculated axial ratio of the molecule was 4. The sedimentation coefficient (s20,w) was 4.92 S at infinite dilution. Comparison of the amino acid composition analysis of avian EF-2 with other mammalian EF-2 molecules as well as EF-G from E. coli showed that there was a striking degree of similarity. The molecular weight calculated from the amino acid residue composition was 81 500. The pI was estimated to be 6.75 by the technique of zone convection electrofocusing. In addition fluorescence and circular dichroism spectra of purified avian EF-2 were analyzed. Circular dichroism spectrum analysis estimated the α-helix content of the molecule to be 19–20%.

A Dispensable Yeast Ribosomal Protein Optimizes Peptidyltransferase Activity and Affects Translocation

Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k(cat)/K(s)) corresponding to the rate of peptide bond formation; this k(cat)/K(s) was lowered 3-fold to 1.15 min(-1) mm(-1) in the L41 mutant compared with 3.46 min(-1) mm(-1) in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 microm EF2 compared with 0.02 microm for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 microm of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 microm in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.