Role of Ribosome Recycling Factor in Translational Coupling as a Ribosome Releasing Factor

ABSTRACT Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

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Ribosome recycling is not critical for translational coupling in Escherichia coli

eLife ◽

10.7554/elife.59974 ◽

2020 ◽

Vol 9 ◽

Author(s):

Kazuki Saito ◽

Rachel Green ◽

Allen R Buskirk

Keyword(s):

Escherichia Coli ◽

Coupling Efficiency ◽

Ribosome Profiling ◽

Ribosome Recycling Factor ◽

Translational Coupling ◽

E Coli ◽

Major Mechanism ◽

Ribosome Density ◽

Reporter Assays ◽

Complete Translation

We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in Escherichia coli. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3′-UTRs. We also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in E. coli. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in E. coli.

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Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5′-processing and translational coupling

Nucleic Acids Research ◽

10.1093/nar/gkr1185 ◽

2011 ◽

Vol 40 (7) ◽

pp. 3152-3158 ◽

Cited By ~ 15

Author(s):

Yuka Adachi ◽

Hiroshi Kuroda ◽

Yasushi Yukawa ◽

Masahiro Sugiura

Keyword(s):

Translational Coupling

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Role of Cis Expression and Translational Coupling in the Assembly of a Functional Drug Pump

The FASEB Journal ◽

10.1096/fasebj.20.4.a517-a ◽

2006 ◽

Vol 20 (4) ◽

Author(s):

Prajakta Pradhan ◽

Parjit Kaur

Keyword(s):

Translational Coupling ◽

Drug Pump

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Structures of the human mitochondrial ribosome recycling complexes reveal distinct mechanisms of recycling and antibiotic resistance

10.1101/2020.12.20.423689 ◽

2020 ◽

Author(s):

Ravi Kiran Koripella ◽

Ayush Deep ◽

Ekansh K. Agrawal ◽

Pooja Keshavan ◽

Nilesh K. Banavali ◽

...

Keyword(s):

Antibiotic Resistance ◽

Conformational Changes ◽

Messenger Rna ◽

Elongation Factor ◽

Ribosome Recycling Factor ◽

Ribosomal Subunits ◽

Sequence Of Events ◽

Mitochondrial Ribosome ◽

Mitochondrial Elongation

AbstractRibosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Mitochondrial ribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt) are two proteins with mitochondria-specific additional sequences that catalyze the recycling step in human mitochondria. We have determined high-resolution cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) in complex with RRFmt, and the mitoribosomal large 39S subunit in complex with both RRFmt and EF-G2mt. In addition, we have captured the structure of a short-lived intermediate state of the 55S•RRFmt•EF-G2mt complex. These structures clarify the role of a mitochondria-specific segment of RRFmt in mitoribosome recycling, identify the structural distinctions between the two isoforms of EF-Gmt that confer their functional specificity, capture recycling-specific conformational changes in the L7/L12 stalk-base region, and suggest a distinct mechanistic sequence of events in mitoribosome recycling. Furthermore, biochemical and structural assessments of the sensitivity of EF-G2mt to the antibiotic fusidic acid reveals that the molecular mechanism of antibiotic resistance for EF-G2mt is markedly different from that exhibited by mitochondrial elongation factor EF-G1mt, suggesting that these two homologous mitochondrial proteins have evolved diversely to negate the effect of a bacterial antibiotics.

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