YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification.     

RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA

Pseudouridine modifications in helix 69 (H69) of 23S ribosomal RNA are highly conserved among all organisms. H69 associates with helix 44 of 16S rRNA to form bridge B2a, which plays a vital role in bridging the two ribosomal subunits and stabilizing the ribosome. The three pseudouridines in H69 were shown earlier to play an important role in 50S subunit assembly and in its association with the 30S subunit. In Escherichia coli, these three modifications are made by the pseudouridine synthase, RluD. Previous work showed that RluD is required for normal ribosomal assembly and function, and that it is the only pseudouridine synthase required for normal growth in E. coli. Here, we show that RluD is far more efficient in modifying H69 in structured 50S subunits, compared to free or synthetic 23S rRNA. Based on this observation, we suggest that pseudouridine modifications in H69 are made late in the assembly of 23S rRNA into mature 50S subunits. This is the first reported observation of a pseudouridine synthase being able to modify a highly structured ribonucleoprotein particle, and it may be an important late step in the maturation of 50S ribosomal subunits.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.     

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m⁷G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m⁴Cm1402 and m⁵C1407. Modification at m⁵C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m⁴Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell''s own indigenous methyltransferases can play an important role in determining resistance levels.     

Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA—but a single modified nucleoside at position 2501 in 23S rRNA—has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine—a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.     

Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE

The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure–function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA–protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7–8 as well as interactions with loop L1–2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis.     

Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus

Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m⁵C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m⁵C967. In contrast to E. coli RsmF, which introduces a single m⁵C1407 modification, T. thermophilus RsmF modifies three positions, generating m⁵C1400 and m⁵C1404 in addition to m⁵C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 Å resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.     

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′-end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), and N^6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m⁶A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m⁶A in noncoding RNAs.     

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407.     

Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.     

YgdE is the 2'-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA

The rRNAs of Escherichia coli contain four 2'- O- methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'- O- methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE . The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits or ribosomes. Nucleotide C2498 is situated within a highly conserved and heavily modified rRNA sequence, and YgdE's activity is influenced by other modification enzymes that target this region. Phylogenetically, YgdE is placed in the cluster of orthologous groups COG2933 together with S -adenosylmethionine-dependent, Rossmann-fold methyltransferases such as the archaeal and eukaryotic RNA-guided fibrillarins. The ygdE gene has been redesignated rlmM for r RNA l arge subunit m ethyltransferase M .     

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.     

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.     

23S rRNA甲基转移酶RrmJ促进细菌50S亚基后期组装

核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控,需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶,能够甲基化修饰核糖体上肽基转移酶中心（peptidyl transferase center, PTC）内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积,因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明,回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善,50S前体累积现象也得到有效缓解。通过共沉淀实验证明,RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S;当加入S-腺苷甲硫氨酸时,该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明,缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明,U2552位点的修饰发生在50S亚基组装晚期特定阶段,这一事件不仅会加速A环的RNA螺旋折叠,另有可能促进附近PTC区域结构成熟。     

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.     

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

The tylosin-resistance methyltransferase RlmA(II) (TlrB) modifies the N-1 position of 23S rRNA nucleotide G748

The methyltransferase RlmA(II) (TlrB) confers resistance to the macrolide antibiotic tylosin in the drug-producing strain Streptomyces fradiae. The resistance conferred by RlmA(II) is highly specific for tylosin, and no resistance is conferred to other macrolide drugs, or to lincosamide and streptogramin B (MLS(B)) drugs that bind to the same region on the bacterial ribosome. In this study, the methylation site of RlmA(II) is identified unambiguously by liquid chromatography/electrospray ionization mass spectrometry as the N-1 position of 23S rRNA nucleotide G748. This position is contacted by the mycinose sugar moiety of tylosin, which is absent from the other drugs. The selective resistance to tylosin conferred by m(1)G748 illustrates how differences in drug structure facilitate the drug fit at the MLS(B)-binding site. This observation is of relevance for the rational design of novel antimicrobials targeting the MLS(B) site, especially if the antimicrobials are to be used against pathogens possessing m(1)G748.     

Conformational change of 23S RNA in 50S ribosome is responsible for translocation in protein synthesis

Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor -G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.