RNA Sequences Associated with Proteins L1, L9, and L5, L18, L25, in Ribonucleoprotein Fragments Isolated from the 50‐S Subunit of <i>Escherichia coli</i> Ribosomes

Branlant, Christiane; Krol, Alain; Sri-Widada, Johanes; Brimacombe, Richard

doi:10.1111/j.1432-1033.1976.tb11039.x

Cited by 46 publications

(23 citation statements)

References 22 publications

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“…The 5' and 3' ends of the 5S RNA (5-7), as well as residues A-39 and U-40 at the loop end of helix III (8), have been localized by immunoelectron microscopy (IEM) on the central protuberance ofthe 50S subunit, in good agreement with the corresponding IEM locations ofproteins L5, L18, and L25, all of which were also found to lie on the central protuberance (9,10). On the other hand, very little information is available relating to possible interactions between 5S and 23S RNA (11).…”

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confidence: 49%

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Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Dontsova

Тишков

Dokudovskaya

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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A DNA fragment containg the Escherichia coUl 5S rDNA sequence linked to a 17 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with ¶7 polymerase using a mixture of [a-32P]UTP and 4-thio-UTP, yielding a transcript in which 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.Interactions and neighborhoods between 5S rRNA and other components of the Escherichia coli 50S ribosomal subunit have been investigated in a number of laboratories by a variety of techniques. From protein binding studies (e.g., refs. 1 and 2) the 5S RNA has long been known to interact with proteins L5, L18, and L25, and the binding sites of these proteins on the 5S molecule have been studied in detail by footprinting methods (e.g., refs. 3 and 4). The 5' and 3' ends of the 5S RNA (5-7), as well as residues A-39 and U-40 at the loop end of helix III (8), have been localized by immunoelectron microscopy (IEM) on the central protuberance ofthe 50S subunit, in good agreement with the corresponding IEM locations ofproteins L5, L18, and L25, all of which were also found to lie on the central protuberance (9, 10). On the other hand, very little information is available relating to possible interactions between 5S and 23S RNA (11).Our laboratories have made extensive use of mRNA analogues containing 4-thiouridine (thio-U) residues, in order to study contacts between mRNA and 16S RNA by photocrosslinking (12-15), and the success of this approach has prompted us to apply the same methodology in the search for contacts between 5S and 23S RNA. Accordingly, a modified 5S RNA was constructed containing a random distribution of thio-U residues in place of normal uridine, and the modified molecule was reconstituted into 50S subunits or 70S ribosomes as a substrate for photocrosslinking. The principal result of the subsequent analysis, which we report here, was the characterization of a high-yield crosslink from the loop end of helix IV of the 5S RNA to a site close to the peptidyltransferase ring of the 23S RNA.MATERIALS AND METHODS Construction of Modified 5S RNA. Plasmid pKK223-3 (Pharmacia), which carries the E. coli 5S rDNA sequence, was cut with HindIII and Ssp I to yield a 500-bp fragment encompassing the 5S rDNA; this fragment was cloned by standard procedures (16) into phage M13mpl8 (...

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confidence: 49%

“…This suggests that the latter helices must fold back toward the central protuberance, a contention which is supported by the crosslinks to proteins L5 and L18 in helix 84 (29). A ribonucleoprotein fragment (11) found to contain 5S RNA, together with L5, L18, and L25 and a segment of 23S RNA, also involves this area of the 23S RNA (Fig. 6).…”

Section: Resultsmentioning

confidence: 99%

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Dontsova

Тишков

Dokudovskaya

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…L9 has been crosslinked to helix 58 in domain III of the 23 S rRNA (Branlant et al, 1976;Brimacombe et al, 1990). This crosslink is inconsistent with data that suggest L9 binds in domain V of the 23 S rRNA and probably only reflects proximity of helix 58 to L9 .…”

Section: Introductionmentioning

confidence: 78%

“…Several lines of evidence suggest that L9 binds the 3' 12 S fragment of the 23 S rRNA in domain V near L1. First, L9 partitions in a ribonucleoprotein fragment with L1 and a 0200 nt RNA fragment located within 550 to 1000 nt of the 3' end of the 23 S rRNA (Branlant et al, 1976). Secondly, using scanning transmission electron microscopy, Tumminia et al (1994) analyzed the conformation of the 23 S rRNA during assembly.…”

Section: Introductionmentioning

confidence: 99%

Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Adamski

Atkins

Gesteland

1996

Journal of Molecular Biology

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“…Yates Li. Since, this 23S RNA region was only partially characterized using the fingerprint technique (6,7,8), it was therefore of great interest to complete ©) IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Escherichia coli 23S ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5′-end region of the L11 operon

1980

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SUMMARYThe results previously obtained upon studying the Ll-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the Li ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E.coli. Three different sequences were observed in E.coli MRE 600. All three sequences differ in two positions relative to the corresponding sequerne in rrnB cistron from E.coli K12.

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RNA Sequences Associated with Proteins L1, L9, and L5, L18, L25, in Ribonucleoprotein Fragments Isolated from the 50‐S Subunit of Escherichia coli Ribosomes

Cited by 46 publications

References 22 publications

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

Characterization of the Escherichia coli 23S ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5′-end region of the L11 operon

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