Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis. This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon. Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA. The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended. Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected. Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons. To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes. This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information. Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression. Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.

The genes for ribosomal proteins IA and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the IA mutation was found to be cold sensitive for growth at 200C, and SOS-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.The interaction of antimicrobial agents with the bacterial ribosome has been a significant area of research for many years because of its relevance to infectious diseases (20, 39). Erythromycin and other macrolide antibiotics in particular have been widely studied for their effects on the functions of the ribosome during translation (12). These compounds bind strongly to the 50S ribosomal subunit of both gram-positive (12, 35) and gram-negative (46) cells and interfere with the elongation of the nascent peptide chain (9, 13,52 (55). Affinity labeling studies with erythromycin derivatives have identified a strong interaction of the drug with protein L22 and weaker associations with proteins L2, IA, and L15 (2). Each of these proteins has been shown to be essential for-reconstitution of the peptidyltransferase activity of the 50S subunit (24). Recent studies have identified the location of several of these proteins at a common region in the 5OS-subunit structure (11).The involvement of rRNA in erythromycin activity has been indicated by a number of recent reports identifying 23S rRNA mutations leading to erythromycin resistance in Escherichia coli (15,18,50) (26,59).Resistance to erythromycin can also result from changes in ribosomal proteins in E. coli (1,(42)(43)(44)(45)58) and in Bacillus species (48,49,56). Whereas the RNA sequence changes have * Corresponding author. Phone: (615) 461-7040. been specifically identified, no sequence information about the alterations in ribosomal proteins leading to erythromycin resistance is presently available. Phenotypic effects of alterations in ribosomal proteins L4 and L22 in two resistant mutants of E. coli were described many years ago (1, 58). We have recently acquired these mutants and have determined the specific DNA sequence c...

Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M1, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.