ABSTRACT
Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life's oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ or Mn2+ could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ and Mn2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+ and Mn2+ were abundant, these findings suggest that Fe2+ and Mn2+ played a role in early ribosomal evolution.
ABSTRACT
Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates â¼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
Subject(s)
Evolution, Molecular , Models, Genetic , Ribosomes/genetics , Magnesium/chemistry , Models, Molecular , Nucleic Acid Conformation , Peptide Fragments/chemistry , Protein Binding , RNA Cleavage , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/genetics , RNA, Ribosomal, 23S/metabolism , Ribonuclease H/chemistry , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Thermus thermophilus/geneticsABSTRACT
Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5' T7 promoter region, and confirmation of the amplified DNA sequence. Complications arise in applications where long, nonnative sequences are desired in the final RNA transcript. Here we describe a ligase-independent method for the preparation of long synthetic DNA templates for in vitro RNA transcription. In Recursive PCR, partially complementary DNA oligonucleotides coding for the RNA sequence of interest are annealed, extended into the full-length double-stranded DNA, and amplified in a single PCR. Long insertions, mutations, or deletions are accommodated prior to in vitro transcription by simple substitution of oligonucleotides.