Báo cáo khoa học: Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the

Suppression of a cold-sensitive mutant initiation factor 1

by alterations in the 23S rRNA maturation region

Jaroslav M. Belotserkovsky, Georgina I. Isak and Leif A. Isaksson

Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden

Introduction

Bacterial protein synthesis, as directed by the action of

the ribosome, can be broadly divided into four main

phases – initiation, elongation, termination, and recy￾cling. Initiation is the rate-limiting step [1]. This phase

is mediated by initiation factor 1 (IF1), initiation fac￾tor 2, and initiation factor 3. IF1 is the smallest of the

initiation factors [2]. Its structure has been determined

by NMR spectroscopy, revealing that IF1 is a member

of the oligomer-binding (OB-fold) family of proteins,

with structural similarities to cold shock proteins [3].

In addition, IF1 has been shown to complement

lesions in several cold shock response proteins in Bacil￾lus subtilis [4]. The interaction of IF1 with the bacterial

ribosome has been investigated by chemical probing

[5], mutagenesis studies [6], and crystallography [7,8].

These data indicate that IF1 makes contacts with the

functionally important bases G530, A1492 and A1493

in 16S rRNA. In addition to its direct involvement in

translation initiation [1], IF1 has been shown to be an

RNA chaperone [9], as well as playing a role in tran￾scriptional antitermination in Escherichia coli [10].

More recently, it has been reported that IF1 acts as a

sensor of cis-elements in mRNA [11] and, together

with initiation factor 3, determines the rates of ribo￾somal subunit joining by inducing conformational

changes in the 30S subunit [12].

Each of the seven rRNA operons in E. coli is tran￾scribed as a single primary transcript. The order of

gene products relative to the start of transcription is

16S, 23S, and 5S, with some tRNA species between

16S and 23S as well as at the end of the transcript. As

transcription proceeds, the rRNA forms secondary

structures that are substrates for the binding of ribo￾somal proteins and maturation factors [13]. Among

these structures are double-stranded stems that are

composed of terminal flanking sequences of 16S and

23S rRNAs. These ‘processing’ stems are substrates for

RNase III, and other ribonucleases that trim the stems

Keywords

Escherichia coli; RNase III; rRNA mutation;

rRNA processing; translation

Correspondence

L. A. Isaksson, Department of Genetics,

Microbiology and Toxicology, Stockholm

University, S-10691 Stockholm, Sweden

Fax: +46 8 164315

Tel: +46 8 164197

E-mail: [email protected]

(Received 24 January 2011, revised 1 March

2011, accepted 14 March 2011)

doi:10.1111/j.1742-4658.2011.08099.x

Genetic selection has been used to isolate second-site suppressors of a

defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia

coli. The suppressor mutants specifically map to a single rRNA operon on a

plasmid in a strain with all chromosomal rRNA operons deleted. Here, we

describe a set of suppressor mutations that are located in the processing stem

of precursor 23S rRNA. These mutations interfere with processing of the 23S

rRNA termini. A lesion of RNase III also suppresses the cold sensitivity.

Our results suggest that the mutant IF1 strain is perturbed at the level of

ribosomal subunit association, and the suppressor mutations partially com￾pensate for this defect by disrupting rRNA maturation. These results support

the notion that IF1 is an RNA chaperone and that translation initiation is

coupled to ribosomal maturation.

Abbreviations

Amp, ampicillin; Cm, chloramphenicol; IF1, initiation factor 1; Kan, kanamycin; Tet, tetracycline; TIR, translation initiation region.

FEBS Journal 278 (2011) 1745–1756 ª 2011 The Authors Journal compilation ª 2011 FEBS 1745