5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

Abstract

In a sequence of 214 nucleotides at the 5' terminus of the Igene mRNA, which codes for the lactose repressor protein of Escherichia coli, (i) an untranslated leader sequence of 28 residues precedes the repressor coding region; (ii) a GUG initiates synthesis of the wild-type repressor; (iii) GUG and AUG are the functional initiators for the synthesis of restart polypeptides activated by early I gene amber mutations, confirming previous assignments for these residues based on protein sequencing data; and (iv) sequences complementary to 16S ribosomal RNA provide stronger potential mRNA-16S rRNA interaction at the wild-type initiation site than at the restart sites. When I mRNA is used to direct the formation of initiation complexes in vitro, ribosomes bind only to the wild-type initiator region.A striking feature of the I mRNA sequence is the presence of a number of in-phase GUGs that have not been observed to serve as initiation signals in vivo in the nonsense mutant strains examined. The selective use of potential initiator triplets in the I mRNA leads to the following conclusions. First, when presented with several neighboring initiator triplets at the wild-type initiator region, ribosomes select the one preceded by the strongest appropriately positioned complementarity to the 16S 31 end. Second, ribosomes do not restart after termination simply by moving to the next available initiator codon. Third, the formation of stable secondary structures predicted for the untranslated I mRNA beyond chain-terminating nonsense mutations may prevent ribosome access to some potential reinitiation sites.The lactose repressor protein, encoded by the lac I gene, exerts negative control over the expression of the lactose operon of Escherichia coli. The possibility of using the I gene to obtain information concerning the specificity of translation initiation was raised several years ago by the discovery that nonsense mutations early in the gene activate reinitiation of repressor synthesis beyond the position of the chain terminating codon. At least three sites within that portion of the mRNA corresponding to the first 62 amino acids of the wild-type repressor protein direct the synthesis of COOH-terminal restart polypeptides which accumulate at 10% of the wild-type repressor level (1-3).The amino acid sequences of the wild-type repressor (4, 5) and the restart polypeptides from amber mutant strains (1-3) predict which I mRNA codons are utilized as reinitiation signals. Whereas restart proteins are not detected in wild-type cells, two restart polypeptides are found in strains carrying amber mutations at amino acid positions 7, 12, and 17. The larger one initiates at a valine codon (presumed to be GUG) in position 23; the smaller one initiates at a methionine codon (AUG) in position 42. When the amber block is moved beyond the first restart

Termination-reinitiation occurs in the translation of mammalian cell mRNAs.

“…Since virtually nothing is known at present about the immediate fate of eucaryotic ribosomes upon termination, we have no basis by which to judge which is the most likely alternative. These are questions which are not yet fully resolved even for bacterial systems, in which termination-reinitiation is a well-documented phenomenon (35,40,41,47,51 pSV2dhfr-gptl3 and -14, however, indicate that the reinitiation process does not necessarily require that the termination event occur upstream of the reinitiation site, only that it not occur too far downstream. Our data do not permit a determination of the maximum distance a ribosome may reach back to reinitiate, only that the process is about 50% efficient over a distance of 50 nucleotides and undetectable at 457 nucleotides.…”

Evidence for endonucleolytic cleavages in decay of lacZ and lacI mRNAs

“…S1 nuclease mapping revealed lacZ mRNA molecules whose 5' and 3' ends were internal to the transcription start and consistent with cleavages at pyrimidine-adenosine bonds 20 to 50 nucleotides apart. With the net 5'-to-3' direction known, lacZ mRNA is probably degraded by sequential cleavages of naked mRNA at vulnerable sites exposed by transit of the last translating ribosome.…”

Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion

“…Early experiments on the genetic code showed that UUG, along with AUG and GUG, can stimulate the binding of N-formylmethionyl-tRNA to ribosomes (3). In vivo experiments have confirmed that UUG acts as an initiator codon in several proteins (1,12,29,37,43). In addition, these proteins all have GGAG-type ribosome binding sites about 4 to 10 nucleotides upstream of the initiation codon, UUG (1,34).…”