Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking

Brandt, Roland; Gualerzi, Claudio O.

doi:10.1016/0014-5793(92)81101-q

Cited by 9 publications

(5 citation statements)

References 27 publications

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“…However, there was no change in translated products, indicating that translational inhibition is specific for rpS3. Although the ribosome filter hypothesis proposes that specific interactions between mRNA and sites on the ribosomal subunit are important for translational control [Brandt and Gualerzi, 1992;Dontsova et al, 1992;Mauro and Edelman, 2002], our results demonstrated that in addition this interaction specifically inhibits its own translation.…”

Section: Discussioncontrasting

confidence: 50%

RpS3 translation is repressed by interaction with its own mRNA

Kim

Joo

et al. 2010

J of Cellular Biochemistry

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Ribosomal protein S3 (RpS3) is a well-known multi-functional protein mainly involved in protein biosynthesis as a member of the small ribosomal subunit. It also plays a role in repairing various DNA damage acting as a repair UV endonuclease. Most of the rpS3 pool is located in the ribosome while the minority exists in free form in the cytoplasm. We here report an additional function of rpS3 in which it represses its own translation by binding to its cognate mRNA. Through RT-PCR of the RNAs co-immunoprecipitated with ectopically expressed rpS3, rpS3 protein was found to interact with various RNAs-endogenous rpS3, 18S rRNA. The S3-C terminal domain was shown to be the major mRNA binding domain of rpS3, independent of the KH domain. This interaction was shown to occur in cytoplasmic fractions rather than ribosomal fractions, and then is involved in its own mRNA translational inhibition by in vitro translation. Furthermore, when Flag-tagged rpS3 was transiently transfected into 293T cells, the level of endogenous rpS3 gradually decreased regardless of transcription. These results suggest that free rpS3 regulates its own translation via a feedback mechanism.

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Section: Discussioncontrasting

confidence: 50%

RpS3 translation is repressed by interaction with its own mRNA

Kim

Joo

et al. 2010

J of Cellular Biochemistry

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“…S18 and S21 are located in the platform region of the 30S subunit that interacts with 5′ ends and the Shine Dalgarno helix in bacterial mRNAs. Specific interaction of this helix and S18 is important for the initiation complex formation. − In the crystal structure of the initiation complex solved by Yusupova et al, the highly flexible and basic N-terminal end of the S18 protein is in close contact with the Shine Dalgarno helix in the initiation complex, whereas in the postinitiation complex, this helix is shifted and rotated to interact with S2 rather than S18 . The S18 protein was determined to be phosphorylated at residues Tyr31, Thr33, and Ser35 (Table ).…”

Section: Resultsmentioning

confidence: 99%

“…Specific interaction of this helix and S18 is important for the initiation complex formation. [42][43][44][45] In the crystal structure of the initiation complex solved by Yusupova et al, the highly flexible and basic N-terminal end of the S18 protein is in close contact with the Shine Dalgarno helix in the initiation complex, whereas in the postinitiation complex, this helix is shifted and rotated to interact with S2 rather than S18. 46 The S18 protein was determined to be phosphorylated at residues Tyr31, Thr33, and Ser35 (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Comprehensive Analysis of Phosphorylated Proteins of Escherichia coli Ribosomes

et al. 2009

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Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.

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“…Cleavage will be induced and the rRNA cleavage sites will be determined using primer extension. Although there have been several crosslinking results reported (Wollenzien et al 1991;Olson et al 1989;Lim et al 1992;Prescott and Goringer 1990;Sundaralingam et al 1975;Dontsova et al 1992;Brandt and Gualerzi 1992;Rinke-Appel et al 1993;Shatsky et al 1991), it is expected that additional cleavage sites will provide a clearer picture of the mRNA position on the ribosome.…”

Section: VImentioning

confidence: 94%

Identification of ribosome–ligand interactions using cleavage reagents

Dj²,

Jm³

et al. 1995

Biochem. Cell Biol.

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To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.

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Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking

Cited by 9 publications

References 27 publications

RpS3 translation is repressed by interaction with its own mRNA

RpS3 translation is repressed by interaction with its own mRNA

Comprehensive Analysis of Phosphorylated Proteins of Escherichia coli Ribosomes

Identification of ribosome–ligand interactions using cleavage reagents

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