Specialized Ribosome System: Preferential Translation of a Single MRNA Species by a Subpopulation of Mutated Ribosomes in Escherichia Coli

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1987 Jul 1

PMID 2440028

Citations 123

Authors

A Hui

H A DE BOER

Affiliations

Soon will be listed here.

Abstract

In Escherichia coli, all mRNAs are translated by one pool of functionally identical ribosomes. Here, we describe a system in which a subpopulation of modified ribosomes are directed to a single mutated mRNA species. This was accomplished by changing the Shine-Dalgarno sequence that precedes the heterologous human growth hormone gene from 5' GGAGG to 5' CCTCC or 5' GTGTG. Translation of these modified mRNAs by wild-type ribosomes is very inefficient. When the anti-Shine-Dalgarno region (i.e., the region complementary to the Shine-Dalgarno sequence) at the 3' end of the gene encoding 16S rRNA (rrnB) was altered from 5' CCTCC to 5' GGAGG or 5' CACAC, thus restoring its potential to base-pair with the mutated human growth hormone mRNA, significant expression of this mRNA occurred. Growth hormone synthesis was dependent on induction of the mutated rrnB operon. Subsequently, these specialized ribosomes were made spectinomycin-resistant by the introduction of a C----U substitution at position 1192 of the 16S rRNA. Thus, host protein synthesis could be shut off by the addition of spectinomycin and the specificity and efficiency of the specialized ribosomes could be assessed. Since the specialized ribosomes represent a nonessential subpopulation in the cell, this system offers an approach to the study of mutations elsewhere in the 16S-rRNA gene that otherwise would be lethal to the cell.

Citing Articles

Autonomous ribosome biogenesis in vitro.

Kosaka Y, Miyawaki Y, Mori M, Aburaya S, Nishizawa C, Chujo T Nat Commun. 2025; 16(1):514.

PMID: 39779722 PMC: 11711502. DOI: 10.1038/s41467-025-55853-7.

Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.

Jann C, Giofre S, Bhattacharjee R, Lemke E Chem Rev. 2024; 124(18):10281-10362.

PMID: 39120726 PMC: 11441406. DOI: 10.1021/acs.chemrev.3c00878.

Differential Selection for Translation Efficiency Shapes Translation Machineries in Bacterial Species.

Farookhi H, Xia X Microorganisms. 2024; 12(4).

PMID: 38674712 PMC: 11052298. DOI: 10.3390/microorganisms12040768.

Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.

Thaenert A, Sevostyanova A, Chung C, Vargas-Rodriguez O, Melnikov S, Soll D Proc Natl Acad Sci U S A. 2024; 121(11):e2321700121.

PMID: 38442159 PMC: 10945757. DOI: 10.1073/pnas.2321700121.

Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus.

Kohl M, Chane-Woon-Ming B, Bahena-Ceron R, Jaramillo-Ponce J, Antoine L, Herrgott L Methods Mol Biol. 2024; 2741:73-100.

PMID: 38217649 DOI: 10.1007/978-1-0716-3565-0_5.

References

DE BOER H, Hui A, Comstock L, Wong E, Vasser M . Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites. DNA. 1983; 2(3):231-5. DOI: 10.1089/dna.1983.2.231. View

Kozak M . Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983; 47(1):1-45. PMC: 281560. DOI: 10.1128/mr.47.1.1-45.1983. View

van Duin J, Ravensbergen C, Doornbos J . Basepairing of oligonucleotides to the 3' end of 16S ribosomal RNA is not stabilized by ribosomal proteins. Nucleic Acids Res. 1984; 12(12):5079-86. PMC: 318901. DOI: 10.1093/nar/12.12.5079. View

Stark M, Gregory R, Gourse R, Thurlow D, Zwieb C, Zimmermann R . Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly. J Mol Biol. 1984; 178(2):303-22. DOI: 10.1016/0022-2836(84)90146-3. View

Gourse R, Takebe Y, Sharrock R, Nomura M . Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc Natl Acad Sci U S A. 1985; 82(4):1069-73. PMC: 397195. DOI: 10.1073/pnas.82.4.1069. View

Jacob W, Santer M, Dahlberg A . A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins. Proc Natl Acad Sci U S A. 1987; 84(14):4757-61. PMC: 305184. DOI: 10.1073/pnas.84.14.4757. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Shine J, Dalgarno L . The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974; 71(4):1342-6. PMC: 388224. DOI: 10.1073/pnas.71.4.1342. View

Steitz J, Jakes K . How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975; 72(12):4734-8. PMC: 388805. DOI: 10.1073/pnas.72.12.4734. View

10.

Dunn J, Studier F . Mutations of bacteriophage T7 that affect initiation of synthesis of the gene 0.3 protein. Proc Natl Acad Sci U S A. 1978; 75(6):2741-5. PMC: 392639. DOI: 10.1073/pnas.75.6.2741. View

11.

Backendorf C, Ravensbergen C, Van Der Plas J, VAN Boom J, Veeneman G, van Duin J . Basepairing potential of the 3' terminus of 16S RNA: dependence on the functional state of the 30S subunit and the presence of protein S21. Nucleic Acids Res. 1981; 9(6):1425-44. PMC: 326767. DOI: 10.1093/nar/9.6.1425. View

12.

Schwartz M, Roa M, Debarbouille M . Mutations that affect lamB gene expression at a posttranscriptional level. Proc Natl Acad Sci U S A. 1981; 78(5):2937-41. PMC: 319474. DOI: 10.1073/pnas.78.5.2937. View

13.

Gold L, Pribnow D, Schneider T, Shinedling S, Singer B, Stormo G . Translational initiation in prokaryotes. Annu Rev Microbiol. 1981; 35:365-403. DOI: 10.1146/annurev.mi.35.100181.002053. View

14.

Singer B, Gold L, Shinedling S, Colkitt M, Hunter L, Pribnow D . Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4. J Mol Biol. 1981; 149(3):405-32. DOI: 10.1016/0022-2836(81)90479-4. View

15.

Van Charldorp R, Van Knippenberg P . Sequence, modified nucleotides and secondary structure at the 3'-end of small ribosomal subunit RNA. Nucleic Acids Res. 1982; 10(4):1149-58. PMC: 320515. DOI: 10.1093/nar/10.4.1149. View

16.

Sigmund C, Ettayebi M, Morgan E . Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of Escherichia coli. Nucleic Acids Res. 1984; 12(11):4653-63. PMC: 318865. DOI: 10.1093/nar/12.11.4653. View