Ribosome Pausing and Stacking During Translation of a Eukaryotic MRNA

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1988 Nov 1

PMID 2850168

Citations 238

Authors

S L Wolin

P Walter

Affiliations

Soon will be listed here.

Abstract

We have devised a sensitive assay to determine the distribution of translating ribosomes on a mRNA. Using this assay to monitor ribosome transit on bovine preprolactin mRNA, we have detected four major positions of ribosome pausing in both wheat-germ and rabbit reticulocyte extracts. Two of these rate-limiting steps represent initiation and termination. One pause occurs after approximately 75 amino acids have been polymerized; signal recognition particle arrests preprolactin synthesis at this position. The other internal pause occurs at 160 amino acids. In these latter two cases ribosomes stop at a GGC glycine codon; however, two other GGC codons are translated without apparent pausing. Surprisingly, we find that up to nine ribosomes are tightly stacked behind each pausing ribosome, such that the ribosome centers are only 27-29 nucleotides apart. The assay should prove useful for probing mechanisms of translational regulation.

Citing Articles

Ribosome profiling reveals differences in global translational vs transcriptional gene expression changes during early biofilm formation.

Mundodi V, Choudhary S, Smith A, Kadosh D Microbiol Spectr. 2025; 13(3):e0219524.

PMID: 39873514 PMC: 11878023. DOI: 10.1128/spectrum.02195-24.

P-stalk ribosomes act as master regulators of cytokine-mediated processes.

Dopler A, Alkan F, Malka Y, van der Kammen R, Hoefakker K, Taranto D Cell. 2024; 187(24):6981-6993.e23.

PMID: 39437780 PMC: 11896023. DOI: 10.1016/j.cell.2024.09.039.

Zika viruses encode 5' upstream open reading frames affecting infection of human brain cells.

Lefevre C, Cook G, Dinan A, Torii S, Stewart H, Gibbons G Nat Commun. 2024; 15(1):8822.

PMID: 39394194 PMC: 11470053. DOI: 10.1038/s41467-024-53085-9.

Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

Tomuro K, Mito M, Toh H, Kawamoto N, Miyake T, Chow S Nat Commun. 2024; 15(1):7061.

PMID: 39187487 PMC: 11347596. DOI: 10.1038/s41467-024-51258-0.

Translation efficiency covariation across cell types is a conserved organizing principle of mammalian transcriptomes.

Liu Y, Rao S, Hoskins I, Geng M, Zhao Q, Chacko J bioRxiv. 2024; .

PMID: 39149359 PMC: 11326257. DOI: 10.1101/2024.08.11.607360.

References

Walter P, Lingappa V . Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986; 2:499-516. DOI: 10.1146/annurev.cb.02.110186.002435. View

PROTZEL A, Morris A . Gel chromatographic analysis of nascent globin chains. Evidence of nonuniform size distribution. J Biol Chem. 1974; 249(14):4594-600. View

Rapoport T, Heinrich R, Walter P, Schulmeister T . Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane. J Mol Biol. 1987; 195(3):621-36. DOI: 10.1016/0022-2836(87)90186-0. View

SIEGEL V, Walter P . Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988; 52(1):39-49. DOI: 10.1016/0092-8674(88)90529-6. View

SIEGEL V, Walter P . The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length. EMBO J. 1988; 7(6):1769-75. PMC: 457167. DOI: 10.1002/j.1460-2075.1988.tb03007.x. View

Gold L . Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988; 57:199-233. DOI: 10.1146/annurev.bi.57.070188.001215. View

Hickey E, Weber L, Baglioni C . Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Proc Natl Acad Sci U S A. 1976; 73(1):19-23. PMC: 335830. DOI: 10.1073/pnas.73.1.19. View

Both G, Furuichi Y, Muthukrishnan S, Shatkin A . Effect of 5'-terminal structure and base composition on polyribonucleotide binding to ribosomes. J Mol Biol. 1976; 104(3):637-58. DOI: 10.1016/0022-2836(76)90126-1. View

Legon S, Robertson H . The binding of 125I-labelled rabbit globin messenger RNA to reticulocyte ribosomes. J Mol Biol. 1976; 106(1):23-36. DOI: 10.1016/0022-2836(76)90298-9. View

10.

Legon S . Characterization of the ribosome-protected regions of 125I-labelled rabbit globin messenger RNA. J Mol Biol. 1976; 106(1):37-53. DOI: 10.1016/0022-2836(76)90299-0. View

11.

Kozak M, Shatkin A . Sequences of two 5'-terminal ribosome-protected fragments from reovirus messenger RNAs. J Mol Biol. 1977; 112(1):75-96. DOI: 10.1016/s0022-2836(77)80157-5. View

12.

Kozak M, Shatkin A . Sequences and properties of two ribosome binding sites from the small size class of reovirus messenger RNA. J Biol Chem. 1977; 252(19):6895-908. View

13.

Legon S, Model P, Robertson H . Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA. Proc Natl Acad Sci U S A. 1977; 74(7):2692-6. PMC: 431248. DOI: 10.1073/pnas.74.7.2692. View

14.

Lazarowitz S, Robertson H . Initiator regions from the small size class of reovirus messenger RNA protected by rabbit reticulocyte ribosomes. J Biol Chem. 1977; 252(21):7842-9. View

15.

Safer B, KEMPER W, Jagus R . Identification of a 48 S preinitiation complex in reticulocyte lysate. J Biol Chem. 1978; 253(10):3384-6. View

16.

Kozak M, Shatkin A . Migration of 40 S ribosomal subunits on messenger RNA in the presence of edeine. J Biol Chem. 1978; 253(18):6568-77. View

17.

von Heijne G, Nilsson L, Blomberg C . Models for mRNA translation: theory versus experiment. Eur J Biochem. 1978; 92(2):397-402. DOI: 10.1111/j.1432-1033.1978.tb12759.x. View

18.

Bergmann J, Lodish H . A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control. J Biol Chem. 1979; 254(23):11927-37. View

19.

Heinrich R, Rapoport T . Mathematical modelling of translation of mRNA in eucaryotes; steady state, time-dependent processes and application to reticulocytes. J Theor Biol. 1980; 86(2):279-313. DOI: 10.1016/0022-5193(80)90008-9. View

20.

Walter P, Blobel G . Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981; 91(2 Pt 1):557-61. PMC: 2111983. DOI: 10.1083/jcb.91.2.557. View