Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Identification and analysis of Escherichia coli ribonuclease E dominant-negative mutants

The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.     

Structure of the Escherichia coli 50 S ribosomal subunit

Freeze-dried and shadowed Escherichia coli 50 S ribosomal subunits have been examined by electron microscopy and a model of the subunit has been constructed. High resolution shadow casting has enabled us to determine independently the absolute hand of the subunit and to reveal some new structural features.     

In vitro synthesis of Escherichia coli ribosomal RNA

Synthesis of ribosomal RNA in a cell-free system was achieved using purified Escherichia coli RNA polymerase and bacterial DNA templates from E. coli, Proteus mirabilis and E. coli/P. mirabilis hybrid strains carrying an E. coli DNA enriched for ribosomal RNA genes.Both direct and indirect competition hybridization revealed that from 5 to 15% of the in vitro product, depending on the template used, had sequences homologous to rRNA. The level of synthesis of sequences homologous to rRNA was related directly to the proportion of rRNA genes in the template. The use of heterologous DNA during competition hybridization ensured at least a 100-fold greater sensitivity for the detection of rRNA sequences than from any messenger RNA sequence.     

The Escherichia coli large ribosomal subunit at 7.5 A resolution

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.     

Pleiotropic effects of ribosomal protein s4 studied in Escherichia coli mutants

Summary A temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.