scholarly journals The Halastavi árva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

2020 ◽  
Author(s):  
Irina S. Abaeva ◽  
Quentin Vicens ◽  
Anthony Bochler ◽  
Heddy Soufari ◽  
Angelita Simonetti ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Internal Ribosomal Entry Sites ◽  
Initiation Mechanism ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Elongation Cycle ◽  
Cryo Electron Microscopy ◽  
A Site ◽  
Sense Codon

ABSTRACTDicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

scholarly journals Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Science ◽  
2015 ◽  
Vol 347 (6217) ◽  
pp. 75-78 ◽  
Author(s):  
Peter S. Shen ◽  
Joseph Park ◽  
Yidan Qin ◽  
Xueming Li ◽  
Krishna Parsawar ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transfer Rna ◽  
Exit Channel ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
New Gene ◽  
Carboxy Terminal ◽  
A Site ◽  
Control Complex

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

scholarly journals The Ccr4-Not complex monitors the translating ribosome for codon optimality

Science ◽  
2020 ◽  
Vol 368 (6488) ◽  
pp. eaay6912 ◽  
Author(s):  
Robert Buschauer ◽  
Yoshitaka Matsuo ◽  
Takato Sugiyama ◽  
Ying-Hsin Chen ◽  
Najwa Alhusaini ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Mrna Decay ◽  
Specific Interaction ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
A Site ◽  
Sense Codon ◽  
Decoding Efficiency

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.

scholarly journals Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp

2005 ◽  
Vol 79 (2) ◽  
pp. 677-683 ◽  
Author(s):  
Randal C. Cevallos ◽  
Peter Sarnow
Keyword(s):  
Internal Ribosome Entry Site ◽  
Intergenic Region ◽  
Rna Virus ◽  
Penaeid Shrimp ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Functional Conservation

ABSTRACT The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

scholarly journals Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

2007 ◽  
Vol 51 (12) ◽  
pp. 4401-4409 ◽  
Author(s):  
Jun-ichi Wachino ◽  
Keigo Shibayama ◽  
Hiroshi Kurokawa ◽  
Kouji Kimura ◽  
Kunikazu Yamane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Recombinant Plasmid ◽  
Coli Strain ◽  
Methyltransferase Activity ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Rrna Molecule ◽  
A Site

ABSTRACT We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

scholarly journals Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components.

1975 ◽  
Vol 67 (3) ◽  
pp. 852-862 ◽  
Author(s):  
G Blobel ◽  
B Dobberstein
Keyword(s):  
Light Chain ◽  
Proteolytic Processing ◽  
Translation Product ◽  
Globin Mrna ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Detergent Treatment ◽  
Processing Activity ◽  
Dog Pancreas

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.

scholarly journals The Ccr4-Not complex monitors the translating ribosome for codon optimality

2019 ◽  
Author(s):  
Robert Buschauer ◽  
Yoshitaka Matsuo ◽  
Ying-Hsin Chen ◽  
Najwa Alhusaini ◽  
Thomas Sweet ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Specific Interaction ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
A Site ◽  
Sense Codon ◽  
Decoding Efficiency ◽  
Insight Into

Control of mRNA decay rate is intimately connected to translation elongation but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

scholarly journals eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

2021 ◽  
Author(s):  
Christopher P. Lapointe ◽  
Rosslyn Grosely ◽  
Masaaki Sokabe ◽  
Carlos Alvarado ◽  
Jinfan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Messenger Rna ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Architectural Framework ◽  
Subunit Joining ◽  
Structural Methods

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

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