Binding Site on R17 RNA for Coat Protein

Spahr, Pierre-François; Farber, M B; Gesteland, Raymond F.

doi:10.1038/222455a0

Cited by 41 publications

(13 citation statements)

References 10 publications

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“…5) is consistent with results from in vivo studies (10). Mutations P53revl and X5O4rev7, which immediately flank the rIIB initiation codon, are slightly less sensitive to regA protein; 5 ,tM regA, which eliminates the ribosome (Fig. 3A).…”

Section: Methodssupporting

confidence: 79%

“…One general strategy for regulating the synthesis of protein from mRNA involves blocking initiation by ribosomes with a repressor protein. Two classic receptors of this type are the bacteriophage T4 gene 32 protein (3,4) and the coat protein encoded by the bacteriophage R17 (5). Ribosome biosynthesis in Escherichia coli is accomplished through similar posttranscriptional regulation (6).…”

mentioning

confidence: 99%

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Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation.

Winter

Morrissey

Gauss

et al. 1987

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

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The bacteriophage T4 regA protein is a translational repressor of a subset of phage mRNAs. We show here that purified regA protein binds specifically to target mRNAs near the initiating AUG and occludes binding of ribosomes. Translational repression by regA protein diminishes expression of many genes whose mRNA sequences around the initiating AUG codons are different. A comparison of nucleotide sequences from several regA-repressed mRNAs suggests that the initiating AUG is an important, but not sufficient, sequence for regA binding.

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Section: Methodssupporting

confidence: 79%

mentioning

confidence: 99%

Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation.

Winter

Morrissey

Gauss

et al. 1987

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

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“…Complex I has been implicated in translational control of the RNA synthetase cistron since addition of coat protein to phage RNA reduces its messenger activity in an in vitro protein-synthesizing system and depresses the synthesis of the RNA synthetase (4)(5)(6)9); it specifically inhibits the initiation step of that synthesis (8,(11)(12)(13). The mechanism by which the coat protein prevents translation of the RNA synthetase cistron is not known.…”

mentioning

confidence: 99%

Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17

Bernardi

Spahr

1972

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

148

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The Coat protein of RNA bacteriophages functions as a repressor of translation of the cistron coding for RNA synthetase (the phage-coded subunit of the RNA replicating enzyme) (1-8).Isolated RNA and coat protein can interact to form two kinds of complexes: Complex I in which a few molar equivalents of coat protein [one (9)-six (10) ] bind to one equivalent of RNA leading to formation of a complex that sediments at the same rate as free RNA, and Complex II in which about 180 coat-protein molecules bind to one molecule of RNA to form a complex that is phage-like, but not infectious (10). Complex I has been implicated in translational control of the RNA synthetase cistron since addition of coat protein to phage RNA reduces its messenger activity in an in vitro protein-synthesizing system and depresses the synthesis of the RNA synthetase (4-6, 9); it specifically inhibits the initiation step of that synthesis (8,(11)(12)(13). The mechanism by which the coat protein prevents translation of the RNA synthetase cistron is not known. We present evidence that the coat protein binds to the initiation site of the RNA synthetase cistron and directly prevents the ribosomes from translating that cistron. We show that when Complex I is formed in vitro and then incubated with Ti RNase, an RNA fragment is protected from nucleolytic degradation; this fragment (59 residues) contains the nucleotide sequence of the region of the R17 RNA preceding the cistron coding for the RNA synthetase and the first two codons of that cistron. The protection by the coat protein of this region from nuclease degradation is specific: when R17 RNA is mixed with the coat protein of Q,3 (an RNA phage serologically unrelated to R17) and the mixture is degraded by T1 RNase, no comparable fragment is found. MATERIAL AND METHODS[3H]Uridine-labeled R17 RNA (2.2 X 104 cpm/,g) and 2P-labeled R17 RNA (3 X 106 cpm/,ug) were prepared as in refs.14 and 15, respectively. Coat proteins of phages R17 and Q0were extracted by the acetic acid procedure (10); the ability of these protein preparations to repress in vitro synthesis of RNA 3033 synthetase was tested (4). Pancreatic RNase was obtained from Worthington and T1 and U2 RNases from Sankyo. Acid RNase and acid phosphatase B from spleen were a gift from Dr. G. Bernardi; they were prepared as described (16,17).Formation of Coat Protein-RNA Complex and Isolation of the Fragment Protected from Nuclease Degradation. The incubation mixture contained in a final volume of 0.6 ml: B2P-labeled R17 RNA (1.15 nmol; 3.2 X 109 cpm), R17 coat protein (4.5 nmol), 100 mM Tris HCl (pH 7.5), 10 mM Mg acetate, and 80 mM KCl (TMK buffer). After 10 min at 00, 0.18 ml of a T1 RNase solution (0.7 mg/ml in 10 mM TrisHCl pH 7.5-2 mM EDTA) were added. The mixture was incubated at 220 for 30 minm then chilled on ice and layered onto two sucrose density gradients (10-20% sucrose in TMK buffer) and centrifuged at 35,000 rpm for 18 hr at 40 in a SW41 Spinco rotor. The gradients were collected at 40 into plastic tubes (27 fractions). Aliq...

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“…B denotes the position of the blue dye marker (xylene cyanole FF) RNA a t a position about 40°/, the way into the molecule from the 5' end. The 40°/, fragment, which sediments a t about 15 S, represents the 5'-terminal 1300-1400 nucleotides of the molecule [18], its yield of terminal pppG residue is nearly equivalent to that of the intact RNA. It can therefore be con- …”

Section: Bmentioning

confidence: 99%