Independent in vitro assembly of all three major morphological parts of the 30S ribosomal subunit of Thermus thermophilus

Abstract

Fragments of the 16S rRNA of Thermus thermophilus representing the 3 H domain (nucleotides 890±1515) and the 5 H domain (nucleotides 1±539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3 H RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3 H domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5 H RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit Keywords: 30S ribosomal subunit; domain structure of ribosomal particles; reconstitution of ribosomal domains; ribosomal proteins; rRNA.The 16S RNA of the small ribosomal subunit is divided into three major domains [1]. Each domain and a defined set of ribosomal proteins are involved in the formation of different morphological parts (lobes) of the subunit. The 5 H domain (nucleotides 1±563) corresponds to the central body of the 30S subunit, the central domain (nucleotides 564±915) is involved in the formation of the side bulge or platform, whereas the 3 H major domain (nucleotides 919±1396) is localized in the head region of the subunit; there is also an additional 3 H minor domain (residues 1397±1542).It has previously been shown that the isolated fragments of the 16S RNA corresponding to its major domains are capable of assembling in vitro with specific sets of ribosomal proteins into compact RNPs. Ofengand and co-workers [2] demonstrated the formation of RNP containing the 5 H domain of Escherichia coli 16S RNA and four ribosomal proteins. Noller and co-workers [3] showed that the fragment of E. coli 16S RNA comprising the 3 H domain assembles with a group of eight ribosomal proteins. The proteins bound in both complexes were the same as those found to be associated with these domains in the intact 30S subunit. Recently we have shown formation of RNP containing the central domain of the 16S RNA from Thermus thermophilus and five ribosomal proteins [4].In this paper we report on the assembly of RNP particles consisting of the 5 H and 3 H domains of the T. thermophilus 16S rRNA and corresponding sets of ribosomal proteins from this bacterium.

Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state

“…These 2°domains are segregated into independent and autonomous three-dimensional domains (3D domains) in the assembled SSU. Each 2°domain of the 16S rRNA folds and assembles with the appropriate ribosomal proteins into a 3D domain, independent of other 2°do-mains (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1999). One 3D domain is called the head and others are called the body and the platform (Brimacombe et al 1983;Wimberly et al 2000).…”

Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria

“…Previous studies have shown that SSUs from thermophilic bacteria could be reconstituted in vitro Agalarov et al 1999), but it was unknown if these bacteria undergo similar temperature-dependent 16S rRNA containing intermediate formation as documented for E. coli. This study demonstrates reconstitution of functional SSUs in vitro from the thermophilic bacteria G. kaustophilus and T. thermophilus and reveals that both these bacteria form at least two distinct temperature-dependent intermediates (Intermediates 1 and 2) during the assembly process.…”

“…These bacteria were chosen due to a drastically different optimum growth temperature from that of E. coli (37°C) and the range in optimal growth temperature (G. kaustophilus 55°C, T. thermophilus 68°C) as well as previous data suggesting that in vitro assembly of SSUs from these organisms is possible , Agalarov et al 1999. Not only do these bacteria differ in the optimal growth temperature, but they also represent diverse morphologic, metabolic, and physiologic groups of bacteria.…”

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

“…All figures containing three-dimensional structures were prepared using the PyMOL and the Protein Data Bank file 2AW7 (Schuwirth et al 2005). domain exhibits similar patterns of dependencies (see Fig. 1A) and can be assembled independently in vitro (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1998Agalarov et al , 1999.…”

“…Also, in both the 59 and 39 major domains Fe(II)-S8 mediated cleavages in 30S subunits were absent in binary complexes. Restoration of cleavage patterns required the assembly of entire domains, highlighting the significance of long-range interactions in assembly, even between independent events (Weitzmann et al 1993;Samaha et al 1994;Agalarov et al 1998Agalarov et al , 1999.…”