ABSTRACT
Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.
Subject(s)
Cryoelectron Microscopy , Ribonuclease, Pancreatic , Ribosomes , Humans , Anticodon/chemistry , Anticodon/genetics , Anticodon/metabolism , Anticodon/ultrastructure , Catalytic Domain , Cytosol/metabolism , Enzyme Activation , Models, Molecular , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , Ribonuclease, Pancreatic/ultrastructure , Ribosomes/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , RNA Cleavage , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Substrate Specificity , Binding Sites , Stress, PhysiologicalABSTRACT
Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the aminoacyl-tRNA-binding site (A site) of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-centre nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise 'latching' of the decoding centre. The resulting closure of the 30S subunit docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and enabling accommodation of the aminoacyl-tRNA. By contrast, near-cognate complexes fail to induce the G530 latch, thus favouring open 30S pre-accommodation intermediates with inactive EF-Tu. This work reveals long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNAs that elucidate the mechanism of accurate decoding.
Subject(s)
Cryoelectron Microscopy , Protein Biosynthesis , Ribosomes/metabolism , Ribosomes/ultrastructure , Anticodon/chemistry , Anticodon/genetics , Anticodon/ultrastructure , Codon/chemistry , Codon/genetics , Codon/ultrastructure , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/ultrastructure , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/ultrastructure , Guanosine Triphosphate/metabolism , Hydrolysis , Models, Molecular , Peptide Elongation Factor Tu/metabolism , Peptide Elongation Factor Tu/ultrastructure , Protein Domains , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA, Ribosomal, 16S/ultrastructure , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer, Amino Acyl/ultrastructure , Ribosome Subunits/chemistry , Ribosome Subunits/metabolism , Ribosome Subunits/ultrastructure , Ribosomes/chemistryABSTRACT
The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA(SufJ), a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA(SufJ) contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL(SufJ) or tRNA(SufJ) does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL(SufJ) and ASL(Thr) bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34-37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL(SufJ) imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA(SufJ) during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.
Subject(s)
Genes, Suppressor , Protein Biosynthesis/genetics , RNA, Ribosomal, 16S/ultrastructure , RNA, Transfer/ultrastructure , Ribosomes/genetics , Anticodon/genetics , Anticodon/ultrastructure , Crystallography, X-Ray , Escherichia coli , Nucleic Acid Conformation , Nucleotides/chemistry , Nucleotides/genetics , RNA, Messenger/genetics , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Transfer/chemistry , RNA, Transfer/genetics , Thermus thermophilus/geneticsABSTRACT
The complex conformational states of the anticodon loop of yeast tRNA(Phe) which we had previously studied with relaxation experiments by monitoring fluorescence of the naturally occurring Wye base, are analyzed using time and polarization resolved fluorescence measurements at varying counterion concentrations. Synchrotron radiation served as excitation for these experiments, which were analyzed using modulating functions and global methods. Three conformations of the anticodon loop are detected, all three occurring in a wide range of counterion concentrations with and without Mg2+, each being identified by its typical lifetime. The fluorescence changes brought about by varying the ion concentrations, previously monitored by steady state fluorimetry and relaxation methods, are changes in the population of these three conformational states, in the sense of an allosteric model, where the effectors are the three ions Mg2+, Na+ and H+. The population of the highly fluorescent M conformer (8ns), most affine to magnesium, is thus enhanced by that ligand, while the total fluorescence decreases as lower pH favors the H+-affine H conformer (0.6ns). Na+-binding of the N conformer (4ns) is responsible for complex fluorescence changes. By iterative simulation of this allosteric model the equilibrium and binding constants are determined. In turn, using these constants to simulate equilibrium fluorescence titrations reproduces the published results.
Subject(s)
Anticodon/ultrastructure , RNA, Transfer, Amino Acid-Specific , RNA, Transfer, Phe , RNA, Transfer/ultrastructure , Saccharomyces cerevisiae/genetics , Fluoroimmunoassay , Guanine/analogs & derivatives , Magnesium/pharmacology , Models, Chemical , Nucleic Acid Conformation , RNA, Transfer, Amino Acid-Specific/metabolism , RNA, Transfer, Phe/metabolism , Spectrometry, FluorescenceABSTRACT
The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.
Subject(s)
Anticodon/metabolism , RNA, Transfer, Amino Acid-Specific/metabolism , RNA, Transfer, Glu/metabolism , RNA, Transfer, Phe/metabolism , RNA, Transfer/metabolism , Thiouridine , Anticodon/ultrastructure , Circular Dichroism , Nucleic Acid Denaturation , RNA, Bacterial/metabolism , RNA, Fungal/metabolismABSTRACT
Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the "L" shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart.
