The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.

Berkhout, Ben; Smit, Maarten H. de; Spanjaard, Remco A.; Blom, T; Duin, Jan van

doi:10.1002/j.1460-2075.1985.tb04082.x

Cited by 38 publications

(27 citation statements)

References 39 publications

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“…This structure acts by blocking ribosome binding to the lysis start; however, it can be physically disrupted by ribosomes reaching the coat gene's stop codon and scanning backward toward the lysis start. In this way, $5% of ribosomes translating the coat gene also translate the lysis gene (Min Jou et al 1972;Kastelein et al 1982;Berkhout et al 1985Berkhout et al , 1987. Less stable versions of the RNA secondary structure yield earlier lysis; more stable versions block lysis expression altogether .…”

Section: Discussionmentioning

confidence: 99%

Genomewide Patterns of Substitution in Adaptively Evolving Populations of the RNA Bacteriophage MS2

Betancourt

2009

Genetics

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Experimental evolution of bacteriophage provides a powerful means of studying the genetics of adaptation, as every substitution contributing to adaptation can be identified and characterized. Here, I use experimental evolution of MS2, an RNA bacteriophage, to study its adaptive response to a novel environment. To this end, three lines of MS2 were adapted to rapid growth and lysis at cold temperature for a minimum of 50 phage generations and subjected to whole-genome sequencing. Using this system, I identified adaptive substitutions, monitored changes in frequency of adaptive mutations through the course of the experiment, and measured the effect on phage growth rate of each substitution. All three lines showed a substantial increase in fitness (a two-to threefold increase in growth rate) due to a modest number of substitutions (three to four). The data show some evidence that the substitutions occurring early in the experiment have larger beneficial effects than later ones, in accordance with the expected diminishing returns relationship between the fitness effects of a mutation and its order of substitution. Patterns of molecular evolution seen here-primarily a paucity of hitchhiking mutations-suggest an abundant supply of beneficial mutations in this system. Nevertheless, some beneficial mutations appear to have been lost, possibly due to accumulation of beneficial mutations on other genetic backgrounds, clonal interference, and negatively epistatic interactions with other beneficial mutations.

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Section: Discussionmentioning

confidence: 99%

Genomewide Patterns of Substitution in Adaptively Evolving Populations of the RNA Bacteriophage MS2

Betancourt

2009

Genetics

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“…The separated proteins were electrophoretically transferred on nitrocellulose by the method of Towbin et al (40). Filters were blocked overnight with 0.05% Tween 20 and incubated with rabbit antiserum against a coat-lysis fusion protein (5 Electron microscopy. Cell suspensions were fixed in 4% formaldehyde in medium for 30 min and layered on a pioloform film-covered grid.…”

Section: Methodsmentioning

confidence: 99%

Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope

et al. 1988

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Bacterial lysis induced by the expression of the cloned lysis gene of the RNA bacteriophage MS2 in Escherichia coli was shown to be under the same regulatory control mechanisms as penicillin-induced lysis. It was controlled by the stringent response and showed the phenomenon of tolerance when E. coli was grown at pH 5. Changes in the fine structure of the murein were found to be the earliest physiological changes in the cell, taking place 10 min before the onset of cellular lysis and inhibition of murein synthesis. Both the average length of the glycan strands and, with a time lag, the degree of cross-linkage were altered, indicating that a lytic transglycosylase and a DD-endopeptidase had been triggered. After extensive separation of the membranes by isopycnic sucrose gradient centrifugation, the lysis protein was present predominantly in the cytoplasmic membrane and in a fraction of intermediate density and, to a lesser degree, in the outer membrane, irrespective of the conditions of growth. However, only under lysis-permissive conditions could a 17% increase in the number of adhesion sites between the inner and outer membranes be observed. Thus, a causal relationship between lysis and the formation of lysis protein-induced adhesion sites seems to exist.Bacteriophages have developed various mechanisms of quite different complexity to escape from their host at a well-controlled time point during their infection cycle. Some of the small phages such as 4iX174 and MS2 have reduced their contribution to this important step to a single lysis gene. These genes code for small-molecular-weight membrane proteins. Their expression has been proven to be sufficient to induce lysis of the host (6,8,14,21,41). However, it is not clear how these proteins function on a molecular level or if host functions are involved as well.The lysis gene (L) of phage MS2 has been cloned, and a temperature-inducible expression system has been constructed (21). Intensive studies of the physiological functions of the host cell after lysis gene induction failed to detect any changes, including the rate of murein synthesis preceding the onset of bacterial lysis (18). The primary effect of the lysis protein, therefore, remains in question.For further elucidation of the mechanism of action of the MS2 lysis protein, its interaction with the bacterial cell wall has been investigated by determining its specific distribution in the bacterial cell wall and its structural effects on both the membranes and the murein sacculus. MATERIALS AND METHODSBacterial strains and plasmids. Escherichia coli M5219 (36) harboring a defective prophage which codes for a temperature-sensitive repressor (cI587) was transformed with either plasmid pMS16 or plasmid pMS20. Both plasmids are pPLa831 derived and contain an MS2 insert downstream of the PL. promoter. The insert in pMS16 is the EcoRI-BamHI segment at positions 869 to 2057 coding for the MS2 coat and lysis genes; the insert in pMS20 is the same as that in pMS16 but carries a small deletion (positions 1764 to 1822)...

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“…It therefore appears that cells may simply tolerate the N-terminal extension of C5, which is dispensable for RNase P activity. This situation is reminiscent of bacteriophage MS2 where the frame for the lysis protein (75 aa) partially overlaps out of phase with the cistron encoding the coat protein (19). The N-terminal 40 aa of the lysis protein, which have little structural order compared with the essential C-terminal domain, were found to be dispensable for function.…”

Section: Role Of the N-terminal Extensionmentioning

confidence: 99%

“…Likewise, most amino acid exchanges between MS2 and related phages fr and R17 occur within these N-terminal 40 aa. It was concluded that the overlap with the coat protein gene is not required for extra coding capacity but serves to couple synthesis of the lysis protein to that of the coat protein (19). Thus, acquisition of nonfunctional N-terminal extensions for the sake of translational coregulation seems to emerge as a principle not only applying to phages, but also to chromosomal genes in bacteria.…”

Section: Role Of the N-terminal Extensionmentioning

confidence: 99%

An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains

Feltens

Gößringer

Willkomm

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis. Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis. Within the sequence encoding the N-terminal extensions and downstream of rpmH, several Thermus species exhibit in-frame deletions͞inser-tions, suggesting relaxed constraints for sequence conservation in this region. Roughly the N-terminal third of T. thermophilus C5 was further shown to be dispensable for RNase P function in vitro by using a precursor tRNA Gly substrate from the same organism. Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.T he ubiquitous enzyme RNase P catalyzes endonucleolytic 5Ј maturation of tRNA primary transcripts in all three domains of life (Archaea, Bacteria, and Eukarya) and in mitochondria and chloroplasts (1, 2). Bacterial RNase P enzymes are composed of a catalytic RNA subunit, Ϸ400 nt in length, and a single small protein of typically 120 aa (3). The RNA subunits alone are catalytically active in vitro but require elevated salt concentrations to compensate for the absence of the protein subunit (4). RNase P holoenzymes are highly efficient catalysts, consistent with their generally low cellular abundance (2).In the majority of bacteria, the rnpA gene encoding the RNase P protein has been identified immediately downstream of the gene for the ribosomal protein L34 (rpmH) and close to the origin of replication oriC (5-7). In Escherichia coli, rpmH and rnpA were shown to be part of the same operon, with two major promoters preceding the rpmH structural gene (7-9). E. coli L34 is produced in excess over the RNase P protein (termed C5), which appears to be regulated at the transcriptional and the translational level. First, three mRNA species derived from the rpmH-rnpA operon were detected, two shorter ones lacking the rnpA cistron and a longer and much less abundant one including it (9). Second, the rnpA codon usage does not correspond to that of highly expressed E. coli genes, such as rpmH (7).We have investigated rnpA expression in thermophilic bacteria of the genus Thermus. This work led to the discovery of an unprecedented mode of (nonviral) gene expression in bacteria: rnpA completely overlaps rpmH in a different reading frame, resulting in the synthesis of unusually long RNase P proteins (163 aa in T. thermophilus). Materials and MethodsFor a detailed description of bacterial strains, cloning procedures, plasmid constructs, and protein and RNA preparation, see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org. Transformation of ...

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The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.

Cited by 38 publications

References 39 publications

Genomewide Patterns of Substitution in Adaptively Evolving Populations of the RNA Bacteriophage MS2

Genomewide Patterns of Substitution in Adaptively Evolving Populations of the RNA Bacteriophage MS2

Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope

An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains

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