Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32

Abstract

Bacteriophage T4 gene 32 lies at the 3' end of a complex transcription unit which includes genes 33, 59, and several open reading frames. In the course of an infection, four major transcripts are synthesized from this unit: two overlapping polycistronic transcripts about 3800 and 2800 nucleotides in length, and two monocistronic gene 32 transcripts about 1150 and 1100 nucleotides in length. These transcripts are made at different times in infection and the polycistronic transcripts have segmental differences in stability. Messenger RNA processing yields a 1025 nucleotide monocistronic gene 32 transcript, and a 135 nucleotide transcript containing part of the gene 59 coding sequence. Processing depends on Escherichia coli encoded ribonuclease E. This pattern of transcription and processing leads to the synthesis of gene 32 mRNA throughout infection, whereas transcripts encoding the upstream genes are present only early in infection. The 3800 nucleotide polycistronic transcript initiates at a promoter that does not require T4 encoded factors for activity. However, full-length synthesis of this transcript depends on the T4 mot gene product. The region upstream of gene 32 also contains four E. coli-like promoters that are active on chimeric plasmids in uninfected cells, but inactive in bacteriophage T4. The location of these cryptic T4 promoters is intriguing in that they lie near the 5' ends of open reading frame B, gene 59 and gene 32. They could play a role in phage development under particular conditions of growth or in bacterial hosts other than those examined here.

Escherichia coli RNase E has a role in the decay of bacteriophage T4 mRNA.

“…RNase E could directly affect T4 mRNA decay by cleaving the messages endonucleolytically, thereby creating sites for the entry of 3'--~ 5' exonucleases, which then processively degrade them. This interpretation is supported by our previous studies of RNase E-dependent mRNA processing at the -71 and -1340 sites in the gene 32 transcription unit (Mudd et al 1988;Carpousis et al 1989), in which the mRNA upstream of the cleavage site was rapidly degraded. In addition, we have recently identified an RNase E-dependent cleavage site RNase E and mRNA decay at position + 831 within the gene 32 message (A.J.…”

“…Figure 4B shows an example of a more complex but more typical T4 transcript pattern. The gene 32 RNA probe hybridizes to polycistronic and monocistronic transcripts, as well as their processed products and decay intermediates (Carpousis et al 1989}. Although it appears that most of the species are significantly more stable in the me-strain, the corn- -(@) cells were infected for 8.5 rain, pulsed with [aH]uridine for 3 rain, and rifampicin and excess cold uridine were added for 3 rain.…”

“…The enzyme is involved in the processing of mRNA from bacteriophage T4 genes 32 and 59 in vivo. Processing of these messages resulted in destabilization of the portion of the mRNA upstream of the cleavage site (Mudd et al 1988;Carpousis et al 1989) and thus may have a role in retroregulation (Schmeissner et al 1984) of upstream gene expression. A comparison of the cleavage sites found in the noncoding RNAs and the T4 mRNAs revealed similarities in sequence at the cleavage sites and in the potential to form RNA secondary structure just downstream of the sites (Tomcs~inyi and Apirion 1985;Mudd et al 1988;Carpousis et al 1989).…”

“…Processing of these messages resulted in destabilization of the portion of the mRNA upstream of the cleavage site (Mudd et al 1988;Carpousis et al 1989) and thus may have a role in retroregulation (Schmeissner et al 1984) of upstream gene expression. A comparison of the cleavage sites found in the noncoding RNAs and the T4 mRNAs revealed similarities in sequence at the cleavage sites and in the potential to form RNA secondary structure just downstream of the sites (Tomcs~inyi and Apirion 1985;Mudd et al 1988;Carpousis et al 1989).As yet, the nucleases that determine the rate of functional decay of total mRNA in E. coil or its phage have not been identified. A mutation in the E. coli ams gene was found to have a five-to sixfold effect on the chemical stability of total E. coli RNAs, but it did not affect functional stability significantly (Kuwano et al 1977;Ono and Kuwano 1979).…”

“…A mutation in the E. coli ams gene was found to have a five-to sixfold effect on the chemical stability of total E. coli RNAs, but it did not affect functional stability significantly (Kuwano et al 1977;Ono and Kuwano 1979). E. coli RNases III (see Portier et al 1987), E (Mudd et al 1988;Carpousis et al 1989), and other as yet unidentified endonucleases (Cannistraro et al 1986; Baga et al 1988; Melefors and von Gabain 1988; Uzan et al 1988) have been implicated in the decay of a few specific bacterial or phage mRNAs. In this paper we present evidence suggesting that E. coli RNase E has a major role in the functional and chemical decay of many bacteriophage T4 mRNAs.…”