Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals.

D A Steege,K C Cone,C Queen,M Rosenberg

doi:10.1016/s0021-9258(18)45429-4

Abstract

The positions of RNA processing events mediated by RNase III and of two ribosome binding sites have been defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL) of bacteriophage lambda. Purified RNase III makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PL start. The positions of these cuts suggest a secondary structure for the RNase III recognition site which is similar to other RNase III sites in which double-stranded cleavage occurs. Structure mapping experiments reveal a pattern of cleavages made in the PL transcript by nucleases specific for single- or double-stranded RNA that support the structure proposed for the RNase III stem and provide clear evidence for the stem-loop formed in the RNA at the position of the N recognition (nutL) site. Our finding that ribosomes bind efficiently in vitro to the region of the PL transcript which includes the AUG codon at position 223 supports other evidence that this triplet is the N protein start codon. The existence of an additional ribosome binding site in the N gene leader region just downstream from the nutL site identifies a second position possibly used for translational initiation or regulation. Its occurrence suggests potential roles for ribosome interaction and/or translation of the leader RNA in regulating phage development and N gene expression.

Highlights

From the $Departmentof Biochemistry, Duke UniversityMedical Center, Durham, North Carolina 27710, IlLaboratory of Biochemistry
The N gene product of bacteriophage X has an essential role in the first step of phage gene expression in suppressing termination in the major leftward (PL)and rightward ( P R ) transcription units[1,2].As a consequence of antitermination mediated by the N protein, transcription extends introegions encoding other key early functions
A 156-nucleotide runoff RNA initiated at the PL promoter was transcribed from DNA digested with T h d, and a 321-nucleotide transcript, from DNA linearized at the HpaI site in the N gene

Summary

Introduction

From the $Departmentof Biochemistry, Duke UniversityMedical Center, Durham, North Carolina 27710, IlLaboratory of Biochemistry. The positions of RNA processing events mediated by RNase I11 and of two ribosome binding sites havebeen defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL)of bacteriophage X. Purified RNase I11 makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PLstart The positions of these cuts suggest a secondary structure for the RNase I11 recognition site which is similar to other RNase I11 sites inwhich double-strandedcleavage occurs. The sequence of the 5’ terminal 149 nucleotides [3], and a mixture of PL and PRtranscripts 100-200 nucleotides long used in ribosome binding experiments yielded only the cro protein initiator from the PRRNA [4].These observations suggested that either the N proteinribosome binding site was weak or not included in RNAs of this length. Current evidence indicates that the latter functions as the N protein initiator, because no internal methionine is found in purified N protein[6],no N - mutations have been found between the two AUGs

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Conclusion