Interactions of translational factor EF-G with the bacterial ribosome before and after mRNA translocation

A conserved translation factor, known as EF-G in bacteria, promotes the translocation of tRNA and mRNA in the ribosome during protein synthesis. Here, EF-G.ribosome complexes in two intermediate states, before and after mRNA translocation, have been probed with hydroxyl radicals generated from free Fe(II)-EDTA. Before mRNA translocation and GTP hydrolysis, EF-G protected a limited set of nucleotides in both subunits of the ribosome from cleavage by hydroxyl radicals. In this state, an extensive set of nucleotides, in the platform and head domains of the 30S subunit and in the L7/L12 stalk region of the 50S subunit, became more exposed to hydroxyl radical attack, suggestive of conformational changes in these domains. Following mRNA translocation, EF-G protected a larger set of nucleotides (23S rRNA helices H43, H44, H89, and H95; 16S rRNA helices h5 and h15). No nucleotide with enhanced reactivity to hydroxyl radicals was detected in this latter state. Both before and after mRNA translocation, EF-G protected identical nucleotides in h5 and h15 of the 30S subunit. These results suggest that h5 and h15 may remain associated with EF-G during the dynamic course of the translocation mechanism. Nucleotides in H43 and H44 of the 50S subunit were protected only after translocation and GTP hydrolysis, suggesting that these helices interact dynamically with EF-G. The effects in H95 suggest that EF-G interacts weakly with H95 before mRNA translocation and strongly and more extensively with this helix following mRNA translocation.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.     

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.     

Probing the phosphates of the Escherichia coli ribosomal 16S RNA in its naked form, in the 30S subunit, and in the 70S ribosome

Ethylnitrosourea is an alkylating reagent which preferentially modifies phosphates in nucleic acids. It was used to map phosphates in naked Escherichia coli 16S rRNA engaged in tertiary interactions through hydrogen bonds or ion coordination. Of the phosphates, 7% are found involved in such interactions, and 57% of them are located in loops or interhelical regions, where they are involved in maintaining local intrinsic structures or long-distance tertiary interactions. The other phosphates (43%) are found in helical regions. These phosphates often occur at the proximity of bulged nucleotides or in irregular helices containing noncanonical base pairs (and bulges) and are assumed to bind cations in order to neutralize negative charges and to stabilize unusual phosphate backbone folding. In the 30S subunit, ENU allowed mapping of phosphates in contact with proteins. The RNA is not uniformly engaged in RNA/protein interactions. Regions 1-51, 250-310, 567-612, 650-670, and 1307-1382 are particularly buried whereas the 3'-terminal domain and the 5'-proximal region (nucleotides 53-218) are exposed. The conformation of 16S rRNA is not drastically affected by protein binding, but conformational adjustments are detected in several defined regions. They are found in the 5' domain (region 147-172), in the central domain (region 827-872), in the 3' major domain (nucleotides 955-956, 994, 1054, 1181, 1257, and 1262-1263), and in the 3'-terminal domain (around 1400). The 50S subunit shields clusters of phosphates located at the subunit interface. The most extensive protections are observed in the 3'-terminal domain (1490-1542), in the central region of the molecule (770-930), and in the upper 3' major domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-hybridization electron microscopy tertiary structure of 16 S rRNA

Seven regions of 16 S rRNA have been located on the surface of the 30 S ribosomal subunit by DNA-hybridization electron microscopy. This information has been incorporated into a model for the tertiary structure of 16 S rRNA, accounting for approximately 40% of the total 16 S rRNA. A structure labeled the platform ring is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500 to 545, and occupies a region on the exterior surface of the subunit near the elongation factor Tu binding site. Ribosomal proteins that have been mapped by immunoelectron microscopy are superimposed onto the model in order to examine possible regions of interaction. Good correlation between the model locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins provide independent support for this model.     

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.