Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA

Abstract

S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. Thus far, S1 has eluded structural determination. We have identified the S1 protein mass in the cryo-electron microscopic map of the Escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1. According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data. Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion. The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit. Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine-Dalgarno sequence, explains the protein's role in the recognition of the 5 region of mRNA. P rotein S1 is the largest ribosomal protein, 68 kDa (1), present in the small subunit of the Escherichia coli 70S ribosome. It has been implicated in the selection of the translation start site on the 30S subunit (2). It has been suggested that a pyrimidinerich region upstream of the Shine-Dalgarno (SD) sequence of mRNA interacts with protein S1 and serves as one of the ribosome recognition sites (3). Protein S1 has been reported to be necessary in some cases for translation initiation (4) and for translation elongation (5). It is the only ribosomal protein that has a high affinity for mRNA (6). As a ribosomal protein, S1 is strikingly atypical. Association of S1 to the ribosome is weak and reversible (ribosome preparations often contain less than stoichiometric amounts of protein S1; ref. 7), whereas most other ribosomal proteins are strongly bound. Furthermore, S1 is an acidic protein, whereas all other ribosomal proteins, with the exception of protein L7͞L12, are basic.The S1 protein consists of 557 amino acid residues. Mild trypsin digestion cleaves S1 into two fragments (8), whereas cleavage with cyanogen bromide yields three different fragments (9). The N-terminal fragment from the latter cleavage contains amino acids 1-193, the middle fragment contains amino acids 224-309, and the C-terminal fragment contains amino acids 332-547 (9). The N-terminal domain binds to the platform region of the 30S subunit (10), whereas the C-terminal domain has been suggested to interact with RNAs (11). S1 has been reported to have an elongated shape in solution (12, 13). Neutron scattering (11) and fluorescence anisotropy experiments (14) indicate that the protein maintains its elongated shape even in its ribosome-bound state.So far, no x-ray crystallographic study has been reported for S1. However, an NMR structure of a 75-aa fragment of S1-type polynucleotide phosphorylase of E. coli, which corresponds to the C-terminal domain of S1, has been determined (15). The structure consists of a five-stranded antiparallel ␤-barrel, a structural motif known...

DgrA is a member of a new family of cyclic diguanosine monophosphate receptors and controls flagellar motor function in Caulobacter crescentus

“…The extragenic suppressor mutation restoring FliL levels was mapped to the coding region of rpsA (ribosomal protein S1). RpsA enhances translation initiation by binding to mRNA regions upstream from the ShineDalgarno sequence and by tethering the mRNAs on the 30S subunit of the ribosome (31)(32)(33). How DgrA and its ligand c-di-GMP modulate FliL levels is a subject for future investigation.…”

Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA

“…Stabilizing the conformation of the downstream pseudoknot (PK2) might prevent putative translational readthrough at the tmRNA-encoded termination codon tmRNA has no obvious SD sequence analogs; for mRNAs lacking an upstream Shine-Dalgarno (SD) sequence, ribosomal protein S1 is required during translation initiation [18]. Based on cryoelectron microscopy, S1 was visualized within the E. coli 30S ribosomal subunit, and the protein is proposed to interact with 11 nucleotides of the mRNA, immediately upstream of the SD sequence [19]. Our data shows protections by the S1 proteins in helix H5 and pseudoknot PK2, suggesting that the interaction between tmRNA and proteins S1 is quite different than that with canonical messenger RNAs.…”

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

“…A matching density corresponding to S21 and S1 was not observed in the reverse map. S21 is a r-protein located between the head and platform and probably not observed because the small size of this protein, S1, when present in the 30S subunit, fills the cleft region between the head and platform (Sengupta et al 2001). However, a corresponding density was not observed in the EM maps of the immature and mature 30S subunits.…”