Purification and conformation of ribosomal protein L25 from E. coli ribosome

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2M LiCl extraction followed by phosphocellulose chromatography in 6 M urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains approximately 16% alpha-helix and approximately 19% beta-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent alpha-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the % alpha-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 M urea used in the preparation of ribosomal proteins.     

Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.     

Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria.

Mammalian genes, when inserted into bacterial plasmid or phage DNAs, will not be expressed into the corresponding specific proteins in E. coli unless proper initiations signals required for recognition by E. coli ribosomes are provided. We have studied these signals and chemically synthesized two DNA duplexes each containing different initiation signals. These have been inserted in front of the Simian virus 40 (SV40) small tumor antigen gene (SV40 t gene) at varying distances from the ATG initiation codon prior to its cloning into pBR322 plasmid DNA. Plasmid containing clones carrying either of these two synthetic ribosome binding sites (RBS) at varying distances from the SV40 t gene all produced a 17K protein identical to authentic t antigen by immunologic, electrophoretic and proteolytic digestion analyses. This provides a novel method to ensure the specific expression of any contiguous mammalian gene to be cloned to bacteria, and also a unique in vivo method for studying the structure-function (efficiency) relationship of RBS with specific base changes.     

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.     

Metabolic regulation of beta-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis.

The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.     

Feedback regulation of ribosomal protein synthesis in E. coli: Localization of the mRNA target sites for repressor action of ribosomal protein L1

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins from a single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.     

Lack of complete cooperativity of ribosome assembly in vitro and its possible relevance to in vivo ribosome assembly and the regulation of ribosomal gene expression.

Earlier studies have shown that the reconstitution of Escherichia coli 50S as well as 30S ribosomal subunits from component rRNA and ribosomal protein (r-protein) molecules in vitro is not completely cooperative and binding of more than one r-protein to a single 16S rRNA (or 23S rRNA) molecule is required to initiate a successful 30S (or 50S) ribosome assembly reaction. We first confirmed this conclusion by carrying out 30S subunit reconstitution in the presence of a constant amount of 16S rRNA together with various amounts of total 30S r-proteins (TP30) and by analyzing the physical state of reconstituted particles rather than by assaying protein synthesizing activity of the particles as was done in the earlier studies. As expected, under conditions of excess rRNA, the efficiency of 30S subunit reconstitution per unit amount of TP30 decreased greatly with the decrease in the ratio of TP30 to rRNA, indicating the lack of complete cooperativity in the assembly reaction. We then asked the question whether the cooperativity of ribosome assembly is complete in vivo. We treated exponentially growing E coli cells with low concentrations of chloramphenicol which is known to inhibit protein synthesis without inhibiting rRNA synthesis, creating conditions of excess synthesis of rRNA relative to r-proteins. Several concentrations of chloramphenicol (ranging from 0.4 to 4.0 micrograms/ml) were used so that inhibition of protein synthesis ranged from 40 to 95%. Under these conditions, we examined the synthesis of RNA, ribosomal proteins and 50S ribosomal subunits as well as the synthesis of total protein. We found that the synthesis of 50S subunits was not inhibited as much as the synthesis of total protein at lower concentrations of chloramphenicol, but the degree of inhibition of 50S subunit synthesis increased sharply with increasing concentrations of chloramphenicol and was in fact greater than the degree of inhibition of total protein synthesis at chloramphenicol concentrations of 2 micrograms/ml or higher. The inhibition of 50S subunit synthesis was significantly greater than the inhibition of r-protein synthesis at all chloramphenicol concentrations examined. These data are consistent with the hypothesis that the cooperativity of ribosome assembly in vivo is also not complete as is the case for in vitro ribosome reconstitution, but are difficult, if not impossible, to explain on the basis of the complete cooperativity model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent (relA+)/relaxed (relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.