SELECTIVE TRANSLATION OF T4 TEMPLATE RNA BY RIBOSOMES FROM T4-INFECTED Escherichia coli

Abstract

Abstract.-The present studies indicate that T4 infection induces an alteration in host ribosomes which restricts the translation of host and other T4-unrelated template RNAs but permits normal translation of T4 RNA. A heat-labile factor has been isolated from T4-infected cell ribosomes which, when combined with normal cell ribosomes, confers upon the latter the property of selective T4 template RNA translation.Recent reports from this laboratory1' 2 and others3 have provided evidence for the coding of transfer RNAs (tRNA) by the T4 genome. The relevance of phage-specific tRNAs to the viral replicative process is not known. Hattman and Hofschneider4 have shown that when Escherichia coli is infected with the RNA phage M12 and then superinfected with T4, progeny M12 phage is strongly inhibited or completely blocked. These investigators further reported that in mixed M12 and T4 infections, M12 protein synthesis is interfered with.5 It therefore seemed possible that T4 tRNAs might be involved in the "shut-off" of RNA phage protein synthesis and perhaps of host protein synthesis.In the course of work to determine the merits of this idea, it was observed that ribosomes from T4-infected cells were much less efficient for translation of MS2 and E. coli template RNAs than were ribosomes from uninfected E. coli, although translation of T4 RNA was similar for both types of ribosomes. This communication describes in vitro studies with normal and T4-infected cell ribosomes and compares their translational efficiency with different RNA templates.Materials and Methods.-Preparation of ribosomes and supernatant fraction: E. coli B cells, normal or 12 min infected with T4am61 (a lysozyme amber mutant, 10 PFU/cell), were ground with alumina (Alcoa 305, 2 gm/gm cells) in the cold, mixed with buffer A (10 mM Tris, pH 7.5, 30 mM KCl, and 10 mM magnesium acetate) plus 5 /g/ml of DNase (RNase-free), and then sedimented at 30,000 X g for 30 min. The S30 supernatant was adjusted to contain 6 mM 2-mercaptoethanol (MSH), centrifuged again as above, incubated at 370 for 1 hr after adding various reagents (ATP, PEP, PEP kinase, all 20 amino acids) as described by Nirenberg et al.,6 dialyzed overnight at 4°against two changes of buffer B (buffer A plus 2 mM MSH), and then centrifuged at 49,000 rpm for 2.5 hr in a Spinco no. 50 rotor. The top three fourths of the clear S165 supernatant was collected and used as the soluble supernatant fraction; the sedimented material was rinsed with buffer B, suspended in the same buffer, and centrifuged at 10,000 X g for 10 min. The nonsedimentable material was used as the source of unwashed ribosomes.

How do bacteria tune translation efficiency?

“…In this Review, “translation efficiency (TE)” is referred to as the rate of protein production per mRNA [14–16]. In other contexts, the same phrase has been defined as the rate of translation elongation, which affects the efficiency with which ribosomes are used [13, 17, 18].…”

Translation of RNA Coliphages by Amino Acid-incorporation Systems of the Enterobacteriaceae

“…Comparisons have been based on translation of very different mRNAs under standard conditions and hence are not absolute. The ribosomes, which are likely responsible for the poor compatibilities, could be modified by phages or episomes (Hsu & Weiss, 1969;Dube & Rudland, 1970;Morrison & Malamy, I y I ) , but as the incidence of poor compatibility is high the differences are more likely inherent. Since the genome of RNA-containing coliphage can sometimes be effectively translated by phylogenetically remote systems (Schwartz, Eisenstadt, Brawerman & Zinder, I 965), translational variations may in part reflect random differences among ribosomes.…”

“…Incompatibilities may be conferred by specific modifying factors (Hsu & Weiss, 1969;Dube & Rudland, 1970;Morrison & Malamy, 1971), or they may also normally exist in polypeptide incorporating systems of Bacillus stearothernzophilus (Lodish, 1970) and a psychrophilic pseudomonad (Szer & Brenowitz, I 970). We have also observed that Streptococcus faecium preparations are unable to translate RNA of the Qp phage of E. coli at high or low Mgt2 levels (Sarimo & Pine, 1971 ;and unpublished experiments).…”

Inhibition of Replication of Ribonucleic Acid Bacteriophage f2 by Superinfection with Bacteriophage T4

“…Infection of Escherichia coli by the deoxyribonucleic acid (DNA) phage T4 results in cessation of host ribonucleic acid (RNA) and protein synthesis. Mechanisms proposed to account for this include alterations in the host membrane (6,8), synthesis of a T4-specific factor for initiation of transcription (30), a loss of certain host transfer RNA (tRNA) species (29) with replacement by phage-specific tRNA species (4,28), and a change in the factors required for initiation of messenger translation (5,16,19,20).…”

“…Several investigators have reported that ribosomes from cultures harvested 12 or 13 min after infection by T4 are inhibited in their ability to translate or bind f2 RNA; experiments mixing components from uninfected and T4-infected cells showed that the origin of the initiation factors determined the ability to recognize f2 RNA (5,16).…”