Protein Folding by Domain V of Escherichia coli 23S rRNA: Specificity of RNA-Protein Interactions

Abstract

The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed.Ribosomes from various sources have shown protein-folding activity in vitro (1,13,14,23). This activity was tested on more than a dozen proteins with various tertiary structures and organizations (1,7,14,20). The activity was found later to be present in the large ribosomal subunit, and on stripping ribosomes of their proteins, it could be assigned to the peptidyl transferase center (PTC) in the larger RNA of the large subunit (8,11,30,35). In Escherichia coli and in other eubacteria in general, the PTC is in domain V (U2016 to G2625 of E. coli) of 23S rRNA.If the same RNA can fold a large number of proteins which are structurally unrelated, the question of specificity arises in the interaction of the unfolded proteins with the RNA. Here we have deciphered the RNA-protein interactions at the nucleotide-amino acid level.To locate the interacting nucleotides of domain V, we used primer extension analysis and determined the positions where the extensions were blocked. We found that the same set of nucleotides interacts with four different proteins tested: bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and chicken egg white lysozyme. Using matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF), we also tested whether the RNA interacted with specific amino acids. In the two proteins tested, BCA and lysozyme, the amino acids involved were specific for each protein and could even be identical between the two proteins. The disposition of the amino acids on the three-dimensional structure shared some common features. The RNA-protein interactions thus appear to have significant specificity. MATERIALS AND METHODSIn vitro synthesis of domain V of E. coli 23S rRNA. The rRNA gene fragment containing domain V was cloned in vector pTZ57R/T (Fermentas) under the control of a T7 promoter. The presence of the domain in selected clones was verified by DNA sequencing. An EcoRI restriction site, just downstream of the domain, was used to linearize the plasmid, and RNA was synthesized using ...

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

“…Binding Sites of Three Protein Substrates on Domain V of 23S rRNA-The protein-binding sites on the central loop and lower part of domain V of 23S rRNA have been reported previously for bovine carbonic anhydrase, lysozyme, malate dehydrogenase, and lactate dehydrogenase protein substrates using UV cross-linking followed by primer extension (21,23). We extended these observations using HCA and dihydrofolate reductase as protein substrates.…”

“…UV Cross-linking-6 M guanidine hydrochloride-denatured proteins (HCA, bovine carbonic anhydrase, and dihydrofolate reductase, all at 30 M) were diluted 100 times in refolding buffer containing domain V of 23S rRNA from E. coli (300 nM), and UV cross-linking was performed immediately in a Bio-Rad GS Gene Linker TM instrument, with 254 nm UV irradiation (600 mJ) (21). For cross-linking with 6AP, 300 nM domain V of 23S rRNA and 0.5 mM 6AP (or 6APi) were mixed and subjected to the same procedure as described above.…”

Involvement of Mitochondrial Ribosomal Proteins in Ribosomal RNA-mediated Protein Folding

“…This interaction is very specific, as only a conserved set of nucleotides bind to polypeptide of any primary sequence (Ref. 28; Fig. 6).…”

“…We have shown earlier that the addition of any other RNA (tRNA, small subunit rRNA, domain II rRNA, etc.) to unfolded BCA or other proteins did not show gel mobility shift upon UV irradiation (17,27,28).…”

Ribosome: The Structure–Function Relation and a New Paradigm to the Protein Folding Problem

“…Since the ribosomes from all sources possess the PTC, having remarkably similar secondary structures, it is not surprising to find that all the PTCs from different sources have protein synthesizing and folding properties which we and others have observed. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] At this point, it should be possible to take the PTC out of the large subunit context and check whether it can fold proteins in vitro. That would make it possible to work out the physico-chemical mechanism of this process.…”