The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.

[1]  M. Springer,et al.  Coexistence of two protein folding states in the crystal structure of ribosomal protein L20 , 2006, EMBO reports.

[2]  M. Springer,et al.  The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control. , 2005, RNA.

[3]  J. Woolford,et al.  The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes. , 2004, Molecular cell.

[4]  J. SantaLucia,et al.  In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA. , 1997, Journal of molecular biology.

[5]  V. Ramakrishnan,et al.  Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution , 1999, Nature.

[6]  Daniel N. Wilson,et al.  Ribosomal Proteins in the Spotlight , 2005, Critical reviews in biochemistry and molecular biology.

[7]  T. Earnest,et al.  Crystal Structure of the Ribosome at 5.5 Å Resolution , 2001, Science.

[8]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[9]  Yan Lin,et al.  DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes , 2008, Nucleic Acids Res..

[10]  E. Brown,et al.  Genetic Interaction Screens with Ordered Overexpression and Deletion Clone Sets Implicate the Escherichia coli GTPase YjeQ in Late Ribosome Biogenesis , 2008, Journal of bacteriology.

[11]  C. Vonrhein,et al.  Structure of the 30S ribosomal subunit , 2000, Nature.

[12]  H. Noller,et al.  Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA. , 1988, Journal of molecular biology.

[13]  A E Dahlberg,et al.  A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. , 1997, Science.

[14]  M. Wahl,et al.  The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control. , 2003, RNA.

[15]  V. Ramakrishnan,et al.  Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA. , 2002, Journal of molecular biology.

[16]  G. Culver,et al.  Interdependencies govern multidomain architecture in ribosomal small subunit assembly. , 2011, RNA.

[17]  H. Noller,et al.  Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA. , 1988, Journal of molecular biology.

[18]  R. Gutell,et al.  Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence. , 1980, Nucleic acids research.

[19]  H. Noller,et al.  Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins. , 1999, RNA.

[20]  H. Noller,et al.  In vitro reconstitution of 30S ribosomal subunits using complete set of recombinant proteins. , 2000, Methods in enzymology.

[21]  R. Green,et al.  Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome. , 2007, Journal of molecular biology.

[22]  S. Liebman,et al.  An accuracy center in the ribosome conserved over 2 billion years. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D B Kell,et al.  Characterization of an autostimulatory substance produced by Escherichia coli. , 2001, Microbiology.

[24]  H. Noller,et al.  Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA. , 1988, Journal of molecular biology.

[25]  H. Noller,et al.  Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. , 1990, Journal of molecular biology.

[26]  L. Gorini,et al.  PHENOTYPIC REPAIR BY STREPTOMYCIN OF DEFECTIVE GENOTYPES IN E. COLI. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Adilakshmi,et al.  Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly , 2008, Nature.

[28]  V. Ramakrishnan,et al.  Selection of tRNA by the Ribosome Requires a Transition from an Open to a Closed Form , 2002, Cell.

[29]  Peter G Schultz,et al.  Systematic chromosomal deletion of bacterial ribosomal protein genes. , 2011, Journal of molecular biology.

[30]  Temple F. Smith,et al.  The origin and evolution of the ribosome , 2008, Biology Direct.

[31]  G. Church,et al.  Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization , 1997, Journal of bacteriology.

[32]  H. Noller,et al.  A functional pseudoknot in 16S ribosomal RNA. , 1991, The EMBO journal.

[33]  G. Siuzdak,et al.  An assembly landscape for the 30S ribosomal subunit , 2005, Nature.

[34]  H. Noller,et al.  Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. , 1986, Journal of molecular biology.

[35]  M. Inouye,et al.  Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli , 2003, Molecular microbiology.

[36]  M. Yusupov,et al.  Crystal Structure of the Eukaryotic Ribosome , 2010, Science.

[37]  L. Iakoucheva,et al.  Intrinsic disorder in cell-signaling and cancer-associated proteins. , 2002, Journal of molecular biology.

[38]  J. P. Rife,et al.  Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA , 2008, Molecular microbiology.

[39]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[40]  V. Uversky,et al.  Why are “natively unfolded” proteins unstructured under physiologic conditions? , 2000, Proteins.

[41]  L. Gorini,et al.  STREPTOMYCIN-INDUCED OVERSUPPRESSION IN E. COLI. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[42]  H. Noller,et al.  Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. , 1995, RNA.

[43]  M. Springer,et al.  The Role of Disordered Ribosomal Protein Extensions in the Early Steps of Eubacterial 50 S Ribosomal Subunit Assembly , 2009, International journal of molecular sciences.

[44]  S. T. Gregory,et al.  Error-prone and error-restrictive mutations affecting ribosomal protein S12. , 2011, Journal of molecular biology.

[45]  Y. Shamoo,et al.  Structure-based analysis of protein-RNA interactions using the program ENTANGLE. , 2001, Journal of molecular biology.

[46]  S. Woodson,et al.  Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly. , 2013, RNA.