gb-2002-3-6-preprint0003 GBJ Deposited research article <p>Identification of attenuation and antitermination regulation in prokaryotes</p> Lathe C Warren III lathe@embl-heidelberg.de Suyama Mikita Bork Peer

EMBL, Meyerhofstr. 1, D69012 Heidelberg, Germany

Max Delbrück Center for Molecular Medicine, D13092 Berlin-Buch, Germany

These authors contributed equally

 Genome Biology 1465-6906 2002 3 6 preprint0003.1 preprint0003.60 http://genomebiology.com/2002/3/6/preprint/0003 This is the first version of this article to be made available publicly, and no other version is available at present. The article was submitted to Genome Biology for peer review. 10.1186/gb-2002-3-6-preprint0003 23 4 2002 30 4 2002 2002 BioMed Central Ltd

Identification of attenuation and antitermination regulation in prokaryotes

The prediction of genes with attenuation or antitermination regulation is reported. The method is based on known characteristics of upstream sequences of regulated genes. We analyzed 26 complete genomes and are thus able to give a more complete picture of attenuation regulation in prokaryotes.

Abstract

Many operons of biochemical pathways in bacterial genomes are regulated by processes called attenuation and antitermination. Though the specific mechanism can be quite different, attenuation and antitermination in these operons have in common the termination of transcription by a RNA 'terminator' fold upstream of the first gene in the operon. In the past, detecting regulation by attenuation or antitermination has often been a long process of experimental trial and error, on a case by case basis. We report here the prediction of over 290 upstream regions of genes with attenuation or antitermination regulation structures in the completed genomes of Bacillis subtilis and Escherichia coli for which extensive experimental studies have been done on attenuation and antitermination regulation. These predictions are based on a computational method devised from characteristics of known terminator fold candidates and benchmark regions of entire genomes. We extend this methodology to 24 additional complete genomes and are thus able to give a more complete picture of attenuation and antitermination regulation in bacteria.

 Bioinformatics Molecular biology Genetics Genome studies

Background

The control of gene expression can occur at many points in the transcription and translation of the genes of bacterial operons. Two mechanisms of operon regulation of great interest are "attenuation" and "antitermination" [1,2,3,4,5]. These mechanisms regulate the early termination of transcription of a wide variety of operons in diverse species. Classically, attenuation occurs when the transcribed RNA upstream of an operon has the ability to fold into two mutually-exclusive RNA-fold structures, one which is termed an antiterminator and the other a terminator. If the terminator hairpin loop is allowed to fold, transcription is ultimately halted. Alternatively, if the antiterminator structure folds, the terminator is precluded from folding and transcription of the operon proceeds. The mechanisms that alternate between these two RNA folds (terminators and antiterminators) are quite diverse. Regulation by antitermination (not to be confused with the alternative antiterminator fold of attenuation) can be differentiated from attenuation by the fact that alteration of the transcription complex (rather than alternate RNA structures) decreases the efficiency of downstream terminators. Though, in reality, the boundary between these two types of regulation is not distinct [3].

Attenuation and antitermination mechanisms have both been described in a wide variety of regulatory and biochemical pathways. These include operons involved in aminoacyl-tRNA biosynthesis, catabolic metabolism, amino-acid biosynthesis, ABC transport systems, ribosomal structural peptides and several others. They have been characterized in genomes as disparate as the low-GC gram-positive Bacillis subtilis and the proteobacteria Escherichia coli. The precise mechanisms that cause the attenuation or antitermination of these operons can be quite distinct. For example, the trp operons of E. coli [4,5] and B. subtilis [6,7], though both regulated by attenuation, are controlled by quite different mechanisms. Other operons, such as the structural ribosomal S10 operon of E. coli [8,9] are regulated by yet a different mechanism. Even between closely related species, the attenuation and antitermination, and upstream regulatory sequences can be entirely different.

Yet, one common and necessary feature of most experimentally described attenuation and antitermination mechanisms is an intrinsic terminator RNA fold structure [2,3]. The stem-loop structure of an intrinsic terminator has been well described [10] and the understanding of the mechanisms of termination has made great progress in recent years [11,12]. This structure is not only found at the location of 'standard' termination of transcription at the end of transcriptional units, but is also, by definition a part of attenuation and antitermination regulation. The major characteristics of this standard terminator structure is that it is relatively short, is energetically stable, has a G/C rich stem, contains a small terminal loop structure and, importantly, also contains a run of U residues on the 3' side of the stem-loop structure [10]. These characteristics of intrinsic terminators have been used in the past to predict terminator structures at the end of transcriptional units (operons) and thus assist in the prediction of transcriptional units in complete genomes [10,13] and on a limited basis for predicting regulatory attenuators [14]. Here we focus on using the characteristics and position of intrinsic terminators to predict and characterize attenuation and antitermination regulation in operons of B. subtilis and E. coli. These mechanisms of regulation are well described in these two genomes. We extend this characterization to an additional 24 genomes representative of the diversity of eubacteria and archeabacteria to give a broader picture of attenuation and antitermination regulation in prokaryotes and in a more automated and extensive manner than previously achieved.

Results

Characterization of attenuators in B. subtilis and E. coli

An extensive literature search for operons in B. subtilis regulated by attenuation or antitermination was conducted and 46 such operons were found. These range from the experimentally well described trp operon to those operons where terminator structures have been found and attenuation is expected though not well characterized experimentally [15,16] (for a full list see http://www.bork.embl-heidelberg.de/Docu/attenuation). These 46 known terminator structures were employed to determine common characteristics of B. subtilis attenuation terminators. Using these characteristics, we screened upstream regions of 3650 B. subtilis genes (using procedures described in Materials and Methods) for terminator folds. Forty-three of the original 46 known terminators found in the literature search were retained in this screening. An additional 1117 upstream folds that fit our criteria were also obtained. In addition, as a control, we used the same filtering and folding methodology on intergenic regions after the sequences were shuffled randomly (952 folds of randomly shuffled sequences were obtained after filtering).

The resulting folds of all intergenic regions and shuffled sequences obtained after filtering were plotted in terms of their stability and length (Figure 1). The known terminator folds lie in a cluster clearly separate and distinct from those folds of randomly shuffled sequences. Terminator folds are of a lesser free energy (ΔG) in relation to length than predicted folds of random sequences. A similar pattern of two easily separated clusters emerges when comparing known terminator structures with folded intragenic regions in which terminator are not expected to be found (data not shown).

<p>Figure 1</p>

Stability and length distributions of stem-loop structures in upstream sequence segments of B. subtilis

Stability and length distributions of stem-loop structures in upstream sequence segments of B. subtilis. The red line shows the largest variance (see Materials and Methods) derived from stem-loop structures in shuffled sequences. Light blue lines give the significance measurements based on standard deviation. The definition for each point together with the orientation of neighboring genes are shown in upper right panel.

Using principal component analysis, we determined the greatest variance of the randomly shuffled sequences. This can give us a measure (using standard deviation) of which folds are significantly different from folds of random sequences (see Materials and Methods). Of the 1160 folds, a total of 203 folds of intergenic regions obtained in our screen fall below the 2nd deviation line (Z ≤ -2) derived from the principal component. These are thus considered significantly different from random folds and possible terminations sites of attenuation or antitermination regulation. Forty-two of these are the known attenuation terminators folds (of the original 43 known folds maintained after filtering). Thus we are able to obtain 91.3% (42/46) of the known and experimentally characterized attenuation and antitermination sites using our filter and significance measure. Additionally, the filter and significance measure screens out over 97.7% (930 of 952) of the folds of random sequences. One hundred and sixty-one (203 total excluding 42 known) folds under the line (Z ≤ -2) are folds not yet analyzed experimentally and could be predicted to be attenuation terminator structures.

A detailed investigation found many of these predictions are strongly supported as a putative attenuation or antitermination sites by genomic context such as the presence of putative promoter sequences, upstream location of putative and known operons, etc. Two terminator structures upstream genes ydbJ and yqhI serve as detailed examples of how genomic context can inform and strongly support the predictions made in Table 1 (Figure 2). Gene ydbJ of B. subtilis is listed as hypothetical with homology to an ABC transporter gene (ATP-binding protein involved in copper transport). The gene immediately downstream, ydbK, has homology to membrane spanning permeases. Using STRING (a search tool for find recurring instances of neighboring genes [17]), orthologs of these two genes are also found in the same order in transcriptional units of 15 other distantly related genomes, suggesting the possibility these genes form an operon. These genes appear to be in a typical ABC transporter operon configuration and several ABC transporter operons are known to be regulated by attenuation in B. subtilis [15,16]. The ydbJ upstream region also has a putative promoter sequence and predicted folds using RNAfold (See Materials and Methods) of the entire upstream sequence suggest it can fold in complex possible antitermination folds (data not shown). Based on this context, we predict this is an ABC transporter operon regulated by attenuation. The second example, yqhI, is the first gene of a run of three genes all having homology to glycine biosynthesis genes in a putative transcriptional unit. This run of three genes also has orthologs found as neighbors in other genomes [17]. Many amino acid biosynthesis operons in B. subtilis are known to be regulated by attenuation [16], thus supporting this prediction.

<p>Figure 2</p>

Schematic drawing of the neighborhood and predicted structures for the B. subtilis genes ydbJ and yqhI

Schematic drawing of the neighborhood and predicted structures for the B. subtilis genes ydbJ and yqhI. Genes are signified by colored arrows and are in orientation of transcription in relation to orientation of reference gene (ydbJ or yqhI). Large blue stem-loop cartoons signify predicted terminator fold in attenuation, 't' is an annotated standard terminator fold. Intergenic regions are drawn to scale and bp lengths of these are given underneath figure.

 <p>Table 1</p>

Predicted attenuators in the genome of B. subtilis

ID

Z-score

Known

Upstream promotera

Geneb

BS0929

-8.895

√

glycerol-3-phosphate dehydrogenase (glpD)

BS2825

-8.473

√

√

acetolactate synthase (acetohydroxy-acid synthase) (large subunit) (ilvB)

BS1310

-6.836

similar to chaperonin (ykkC)

BS0983

-6.733

√

hypothetical protein (yhaW)

BS3920

-6.468

√

√

PTS beta-glucoside-specific enzyme IIABC component (bglP)

BS1257

-6.332

RNA polymerase PBSX sigma factor-like (xpf)

BS2733

-6.238

√

alanyl-tRNA synthetase (alaS)

BS0013

-6.126

√

√

seryl-tRNA synthetase (serS)

BS2184

-5.942

√

hypothetical protein (yphP)

BS3966

-5.840

√

hypothetical protein (iolD)

BS2749

-5.839

√

histidyl-tRNA synthetase (hisS)

BS2520

-5.815

√

√

glycyl-tRNA synthetase (alpha subunit) (glyQ)

BS3396

-5.767

√

√

similar to amino acid permease (yvbW)

BS0215

-5.526

√

√

glycerol-3-phosphate permease (glpT)

BS2255

-5.344

hypothetical protein (ypiA)

BS2600

-5.342

similar to phage-related protein (yqbK)

BS1544

-5.239

√

√

isoleucyl-tRNA synthetase (ileS)

BS3710

-5.196

√

CTP synthetase (ctrA)

BS3895

-5.191

√

similar to pyrimidine nucleoside transport protein (yxjA)

BS3798

-5.164

√

√

PTS sucrose-specific enzyme IIBC component (sacP)

BS2614

-5.152

√

hypothetical protein (yqaQ)

BS2484

-5.148

√

similar to 5-formyltetrahydrofolate cyclo-ligase (yqgN)

BS1319

-5.124

√

√

cobalamin-independent methionine synthase (metC)

BS2682

-4.990

√

hypothetical protein (yraL)

BS3750

-4.958

√

√

threonyl-tRNA synthetase (thrZ)

BS0183

-4.938

√

NADH dehydrogenase (subunit 5) (ndhF)

BS3440

-4.890

√

√

levansucrase (sacB)

BS2715

-4.841

√

similar to formate dehydrogenase (yrhE)

BS2204

-4.819

√

√

xanthine phosphoribosyltransferase (xpt)

BS2139

-4.756

√

√

similar to N-acetylmuramoyl-L-alanine amidase (yomC)

BS2803

-4.750

√

√

valyl-tRNA synthetase (valS)

BS0093

-4.747

√

√

serine acetyltransferase (cysE)

BS3877

-4.696

√

hypothetical protein (yxkD)

BS1357

-4.680

√

√

hypothetical protein (ykrT)

BS0178

-4.675

√

L-glutamine-D-fructose-6-phosphate amidotransferase (glmS)

BS1658

-4.644

√

DNA polymerase III (alpha subunit) (polC)

BS3894

-4.630

√

hypothetical protein (yxjB)

tRNA-Arg

-4.609

tRNA-Arg (trnJ-Arg)

BS0535

-4.505

similar to antibiotic resistance protein (ydfB)

BS3839

-4.472

√

tyrosyl-tRNA synthetase (tyrZ)

BS2961

-4.430

√

√

tyrosyl-tRNA synthetase (tyrS)

BS0441

-4.399

hypothetical protein (ydbB)

BS1143

-4.382

√

tryptophanyl-tRNA synthetase (trpS)

BS1737

-4.375

√

similar to ribonucleoprotein (ymaA)

BS0254

-4.309

√

√

hypothetical protein (yczA)

BS1971

-4.297

√

similar to adenosylmethionine-8-amino-7-oxononanoate aminotransferase (yodT)

BS0643

-4.279

√

√

phosphoribosylaminoimidazole carboxylase I (purE)

BS3026

-4.270

√

√

leucyl-tRNA synthetase (leuS)

BS2889

-4.249

√

threonyl-tRNA synthetase (thrS)

BS3690

-4.191

√

hypothetical protein (ywlC)

BS1188

-4.176

√

√

similar to cystathionine gamma-synthase (yjcI)

BS0521

-4.158

√

hypothetical protein (ydeI)

BS2452

-4.143

√

similar to aminomethyltransferase (yqhI)

BS3604

-4.128

√

hypothetical protein (ywrE)

BS1313

-4.086

√

gamma-glutamyl kinase (proB)

BS3269

-3.989

√

√

similar to ABC transporter (ATP-binding protein) (yusC)

BS0423

-3.974

√

hypothetical protein (ydaH)

BS2504

-3.896

√

similar to transcriptional regulator (Fur family) (yqfV)

BS3900

-3.884

√

√

endo-beta-1,3-1,4 glucanase (bglS)

BS0038

-3.883

√

√

methionyl-tRNA synthetase (metS)

BS2319

-3.879

√

hypothetical protein (ypuF)

BS1683

-3.845

similar to phage-related protein (ymfE)

BS1881

-3.805

√

phosphoenolpyruvate synthase (pps)

BS3513

-3.805

√

putative membrane protein (csbA)

BS0432

-3.796

hypothetical protein (ydaO)

BS3355

-3.795

hypothetical protein (yvaI)

BS2858

-3.784

√

√

phenylalanyl-tRNA synthetase (alpha subunit) (pheS)

BS3668

-3.780

√

hypothetical protein (ywmD)

BS0927

-3.776

√

glycerol uptake facilitator (glpF)

BS0281

-3.739

√

hypothetical protein (ycdC)

BS2306

-3.714

RNA polymerase ECF-type sigma factor (sigma-X) (sigX)

BS0143

-3.709

√

RNA polymerase (alpha subunit) (rpoA)

BS0449

-3.687

√

similar to ABC transporter (ATP-binding protein) (ydbJ)

BS1094

-3.668

√

hypothetical protein (yitC)

BS1370

-3.645

√

motility protein A (motA)

BS3785

-3.618

√

hypothetical protein (ywdL)

BS0104

-3.612

√

ribosomal protein L10 (BL5) (rplJ)

BS3313

-3.584

similar to iron-binding protein (yvrC)

BS0716

-3.572

√

hypothetical protein (yetH)

BS1889

-3.524

√

response regulator aspartate phosphatase (rapK)

BS0673

-3.492

hypothetical protein (yerQ)

BS0002

-3.480

DNA polymerase III (beta subunit) (dnaN)

BS0880

-3.473

√

transcriptional regulator (senS)

BS1548

-3.473

√

√

transcriptional attenuator and uracil phosphoribosyltransferase activity (minor) (pyrR)

BS1409

-3.467

√

hypothetical protein (ykuH)

BS2881

-3.464

initiation factor IF-3 (infC)

BS1651

-3.412

uridylate kinase (smbA)

BS3888

-3.412

√

√

hypothetical protein (yxjH)

BS3889

-3.412

√

√

hypothetical protein (yxjG)

BS0925

-3.407

√

similar to adenosylmethionine-8-amino-7-oxononanoate aminotransferase (yhxA)

BS1205

-3.351

hypothetical protein (yjdG)

BS3784

-3.335

hypothetical protein (spsA)

BS3093

-3.325

√

hypothetical protein (yuaJ)

BS1470

-3.265

√

hypothetical protein (yktD)

BS1549

-3.265

√

uracil permease (pyrP)

BS2841

-3.262

√

aspartokinase II (lysC)

BS2903

-3.222

DNA polymerase I (polA)

BS3446

-3.202

√

hypothetical protein (yvdQ)

BS2984

-3.202

hypothetical protein (ytmQ)

BS1920

-3.201

√

similar to ATP-dependent DNA helicase (yocI)

BS3343

-3.192

√

hypothetical protein (yvgV)

BS3731

-3.192

√

hypothetical protein (ywiA)

BS3278

-3.188

similar to 3-hydroxyacyl-CoA dehydrogenase (yusL)

BS3412

-3.180

transcriptional regulator (LacI family) (lacR)

BS2278

-3.156

hypothetical protein (yphE)

BS3914

-3.141

√

hypothetical protein (yxiF)

BS1550

-3.120

√

√

aspartate carbamoyltransferase (pyrB)

BS0878

-3.120

√

thiamin biosynthesis protein (thiA)

BS2775

-3.111

√

similar to sodium/proton-dependent alanine carrier protein (yrbD)

BS1673

-3.111

dipicolinate synthase subunit A (spoVFA)

BS2569

-3.086

√

site-specific DNA recombinase (spoIVCA)

BS2713

-3.075

√

similar to formate dehydrogenase (yrhG)

BS2539

-3.075

heat-shock protein (dnaJ)

BS3719

-3.068

similar to cardiolipin synthetase (ywiE)

BS2711

-3.061

√

similar to methyltransferase (yrhH)

BS1166

-3.059

√

transcriptional regulator (tenA)

BS1446

-3.057

aminopeptidase (ampS)

BS0503

-3.033

√

hypothetical protein (yddM)

BS1455

-3.014

√

hypothetical protein (ykzG)

BS3164

-2.969

√

transcriptional regulator (comQ)

BS1360

-2.966

√

√

similar to ribulose-bisphosphate carboxylase (ykrW)

BS1536

-2.942

√

similar to acetylornithine deacetylase (ylmB)

BS3133

-2.940

√

similar to polyribonucleotide nucleotidyltransferase (yugI)

BS3635

-2.930

√

flagellar basal-body rod protein (flhO)

BS1659

-2.908

√

hypothetical protein (ylxS)

BS2216

-2.896

hypothetical protein (ypsA)

BS0115

-2.894

√

ribosomal protein S10 (BS13) (rpsJ)

BS0637

-2.878

√

hypothetical protein (yebB)

BS2627

-2.867

similar to transcriptional regulator (phage-related) (Xre family) (yqaE)

BS0745

-2.856

√

similar to quinone oxidoreductase (yfmJ)

BS1785

-2.852

transcriptional regulator (lexA)

BS1737

-2.805

√

similar to ribonucleoprotein (ymaA)

BS1525

-2.800

√

cell-division initiation protein (divIB)

BS2138

-2.782

hypothetical protein (yomD)

BS2162

-2.776

√

hypothetical protein (yokC)

BS1335

-2.740

√

similar to transcriptional regulator (MarR family) (ykoM)

BS0241

-2.658

√

similar to histidine permease (ybgF)

BS2715

-2.649

√

similar to formate dehydrogenase (yrhE)

BS1855

-2.649

√

√

similar to phosphoglycerate dehydrogenase (yoaD)

BS2986

-2.643

√

hypothetical protein (ytmP)

BS1321

-2.642

hypothetical protein (ispU)

BS0386

-2.640

√

similar to transcriptional regulator (TetR/AcrR family) (ycnC)

BS2425

-2.616

similar to exodeoxyribonuclease VII (large subunit) (yqiB)

BS0057

-2.589

similar to amino acid transporter (yabM)

BS3957

-2.586

similar to ABC transporter (ATP-binding protein) (yxdL)

BS1003

-2.585

√

Hit-like protein (hit)

BS3350

-2.580

hypothetical protein (yvaC)

BS1659

-2.573

√

hypothetical protein (ylxS)

BS4050

-2.567

√

hypothetical protein (yybP)

BS2617

-2.559

√

hypothetical protein (yqaN)

BS0806

-2.555

√

acetoin dehydrogenase E1 component (TPP-dependent alpha subunit) (acoA)

BS0636

-2.553

√

GMP synthetase (guaA)

BS1871

-2.546

√

hypothetical protein (yoaS)

BS2152

-2.537

√

hypothetical protein (yolA)

BS1490

-2.535

√

cytochrome caa3 oxidase (subunit II) (ctaC)

BS3210

-2.518

transcriptional regulator (paiA)

BS0558

-2.516

√

hypothetical protein (ydgC)

BS1585

-2.516

similar to phosphoglycerate dehydrogenase (yloW)

BS3625

-2.513

transcriptional regulator (DeoR family) (glcR)

BS2512

-2.483

√

hypothetical protein (yqfN)

BS0164

-2.480

similar to transcriptional regulator (AraC/XylS family) (ybbB)

BS2895

-2.471

√

hypothetical protein (ytcF)

BS2632

-2.456

√

hypothetical protein (yrkS)

BS0804

-2.447

√

hypothetical protein (yfjM)

BS0632

-2.404

√

similar to cation efflux system membrane protein (yeaB)

BS2632

-2.392

√

hypotheitcal protein (yrkS)

BS2713

-2.387

√

similar to formate dehydrogenase (yrhG)

BS0561

-2.386

√

ATP-binding transport protein (expZ)

BS1373

-2.372

√

hypothetical protein (ykvJ)

BS0888

-2.371

√

hypothetical protein (ygaO)

BS2239

-2.369

ketopantoate hydroxymethyltransferase (panB)

BS2746

-2.356

√

hypothetical protein (yrvN)

BS3568

-2.347

UDP-glucose:polyglycerol phosphate glucosyltransferase (tagE)

BS4087

-2.342

√

similar to formate dehydrogenase (yyaE)

BS0745

-2.309

√

similar to quinone oxidoreductase (yfmJ)

BS3473

-2.308

√

similar to mutator MutT protein (yvcI)

BS2596

-2.308

√

similar to phage-related protein (yqbN)

BS1319

-2.296

√

cobalamin-independent methionine synthase (metC)

BS1472

-2.294

√

hypothetical protein (ylaA)

BS3265

-2.287

√

similar to ABC transporter (ATP-binding protein) (yurY)

BS3666

-2.250

required for formate dehydrogenase activity (narQ)

BS2225

-2.236

√

hypothetical protein (yppD)

BS3000

-2.232

√

hypothetical protein (ytfP)

BS1181

-2.230

√

hypothetical protein (yjcB)

BS1735

-2.220

hypothetical protein (ymzC)

BS0668

-2.210

√

hypothetical protein (yerL)

BS1065

-2.204

similar to DNA exonuclease (yirY)

BS3199

-2.201

√

hypothetical protein (yuiF)

BS1302

-2.182

hypothetical protein (ykgB)

BS2921

-2.181

hypothetical protein (ytoI)

BS3142

-2.171

√

similar to multidrug-efflux transporter (yuxJ)

BS2729

-2.163

√

similar to caffeoyl-CoA O-methyltransferase (yrrM)

BS0313

-2.157

hypothetical protein (ycgI)

BS3278

-2.157

similar to 3-hydroxyacyl-CoA dehydrogenase (yusL)

BS1386

-2.151

√

similar to heavy metal-transporting ATPase (ykvW)

BS0245

-2.139

√

similar to two-component sensor histidine kinase [YcbB] (ycbA