Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements 1 1 Edited by M. Gottesman

Stump, Mark; Madison‐Antenucci, Susan; Kokoska, Robert J.; Steege, Deborah A.

doi:10.1006/jmbi.1996.0766

Cited by 4 publications

(6 citation statements)

References 43 publications

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“…Bacteria were collected by centrifugation 2 h after infection and resuspended in 4 ml of a mixture of 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0), and 4 mg of lysozyme per ml per 200-ml culture. IKe replicative-form (RFI) DNA was prepared by the method of Birnboim and Doly (1) with modifications described previously (49). Following resuspension in 2 ml of 10 mM Tris-HCl (pH 7.5)-1 mM EDTA, the DNA was digested with BamHI to linearize contaminating N3 DNA but leave RFI DNA intact.…”

Section: Methodsmentioning

confidence: 99%

“…The f1 mRNA diagram is the work of many authors (29). The IKe mRNAs, as recently defined, range in length from 470 to ϳ2,000 nt (49). Coding regions are indicated by vertical lines.…”

Section: Fig 1 Major F1 and Ike Mrnas And Junctions Between Genes Vmentioning

confidence: 99%

“…Genes V and VII are immediately adjacent on a series of overlapping mRNAs (Fig. 1) that are very similar but not identical to the f1 mRNAs (49), making it likely that any control over pVII synthesis operates at the translational level. A previous study of IKe pV indicated that at least 10 5 copies are made per cell (32), but pVII levels were not determined.…”

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confidence: 99%

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Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe

Madison‐Antenucci

Steege

1998

J Bacteriol

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Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it. Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from Escherichia coli. The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs. Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V. The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation. The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, ∼75-fold. As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred.

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Section: Methodsmentioning

confidence: 99%

“…The f1 mRNA diagram is the work of many authors (29). The IKe mRNAs, as recently defined, range in length from 470 to ϳ2,000 nt (49). Coding regions are indicated by vertical lines.…”

Section: Fig 1 Major F1 and Ike Mrnas And Junctions Between Genes Vmentioning

confidence: 99%

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Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe

Madison‐Antenucci

Steege

1998

J Bacteriol

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“…Genes V and VII are immediately adjacent (Fig. 1A and B), and are expressed on a very similar series of mRNAs (Cashman et al ., 1980; Stump et al ., 1997). They encode proteins used in very different amounts: pV coats the large number of single‐stranded viral DNA molecules before phage assembly, whereas pVII is present in only three to five copies on one tip of the phage (reviewed by Model and Russel, 1988; Russel, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…A. In the mRNA diagram (Stump et al ., 1997), primary transcripts are indicated by asterisks, and the scale in nt is shown. The other mRNAs are the products of post‐transcriptional processing.…”

Section: Introductionmentioning

confidence: 99%

Translation at higher than an optimal level interferes with coupling at an intercistronic junction

Madison‐Antenucci

Steege³

2001

Molecular Microbiology

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In pairs of adjacent genes co‐transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single‐stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild‐type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine–Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

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Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli

Goodrich

Steege

1999

RNA

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To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.

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Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements 1 1 Edited by M. Gottesman

Cited by 4 publications

References 43 publications

Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe

Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe

Translation at higher than an optimal level interferes with coupling at an intercistronic junction

Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli

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