Emory 1992 E.coli

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10.1101/gad.6.1.135Access the most recent version at doi: 1992 6: 135-148Genes Dev.

S A Emory, P Bouvet and J G Belasco Escherichia coli.A 5'-terminal stem-loop structure can stabilize mRNA in

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A 5'-terminal stem-loop structure can

stabilize mRNA in Escherichia coli

S h e r i A . E m o r y / P h i l i p p e B o u v e t / ' ^ a n d J o e l G . B e l a s c o ^ ' ^

^Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 USA;

^Laboratoire de Biologic et Genetique du Developpement, Universite de Rennes I, 35042 Rennes Cedex, France

The 5'-untranslated region of the long-lived Escherichia coli ompA transcript function s as an mR NA stabilizer

capable of prolonging the lifetime in

E. coli

of a num ber of heterolo gous m essag es to whic h it is fused. T o

elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA

5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational

analysis . The presence of a stem-loop no more than 2-4 nucleot ides from the extreme 5 ' terminus of this

RNA segment is crucia l to it s stabil iz ing inf luence, whereas the sequence of the stem-loop is relat ively

unimportant. The potential to form a hairpin very close to the 5' end is a feature common to a number of

stable prokaryotic messages. Moreover, the lifetime of a normally labile message {bla mRNA) can be

prolonged in £. coli by adding a simple hairpin structure at its 5' terminus. Accelerated degradation of ompA

mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end. We propose that

E. coli m essag es beginning wit h a single-stranded RNA segm ent of significant length are preferentially

targeted by a degradative tibonuclease that interacts with the mRNA 5' terminus before cleaving internally at

one or more distal sites.

[Key Words: mR NA s tab i l i ty ; 5 ' - u n t r an s la ted r eg io n ; Escherichia coli; g en e r eg u la t io n ; ompA; R N A s t ru c tu r e ]

Received October 18, 1991; revised version accepted November 27, 1991.

Degradation of mRNA is a cel lu lar process that is h ighly

impor tant for contro ll ing gene express ion . The capacity

to degrade mRNA is essential to the ab il i ty of cel ls to

al ter their patterns of pro tein synthesis rap id ly in re-

sponse to their changing needs. In addit ion , the s teady-

s ta te ce l lu la r co n cen t r a t io n o f a co n t in u o u s ly sy n th e-

s ized me ssage is d irect ly propor t i onal to i ts half - l ife . T he

lifet imes of indiv idual messages can vary widely with in

a s ingle cel l . For ins tance, mRNA half - l ives in Escherich-

ia coh

range f rom seconds to near ly an hour , with an

average l ifet ime of 2-4 min (Pedersen et a l . 1978; Nils-

son et a l . 1984; Donovan and Kushner 1986; Emory and

B elasco 19 9 0) . Desp i te th e imp o r tan ce of mR N A in s ta -

b i l i ty to g en e r eg u la t io n , th e mech an isms an d s t ru c tu r a l

d e te rmin an ts o f p ro k ary o t ic mR NA d ecay a r e n o t we l l

unders tood (Kennell 1986; Belasco and Higgins 1988) .

Elements at the 5 ' and 3 ' ends of bacter ial and phage

messag es h av e b een imp l ica ted in mR NA s tab i l iza t io n .

M o s t p ro k a r y o t i c m R N A s e n d i n a 3 ' - t e r m i n a l s t e m -

loop s tructure, which serves as a pro tective barr ier

against degradation by 3 ' exor ibonucleases (Belasco et a l .

1985; Mott et a l . 1985; Newbury et a l . 1987; Chen et a l .

1988).

Ho wev er , f o r b ac te r ia l mR NAs th a t en d wi th a 3 '

hairp in , there is hard ly any evidence to indicate that s ta-

bility differences in vivo result from differential rates of

ex o n u c leo ly t ic p en e t r a t io n o f th ese 3 ' s tem- lo o p s t ru c-

^Corresponding author.

tu res (Belasco et a l . 1986; Chen et a l . 1988; Chen and

Belasco 1990) . Ins tead , i t appears that the d isparate l i fe-

t imes o f mo s t p ro k ary o t ic mR NAs a r e co n t ro l led b y d e-

cay d e te rmin an ts lo ca ted u p s t r eam o f th e 3 ' en d . Esp e-

c ia l ly n o te wo r t h y a r e a smal l n u m b er o f 5 ' - t e rm in a l

mR NA seg men ts th a t h av e b een sh o wn to s tab i l ize h e t -

ero logous messages to which they are fused . These 5 '

mR NA s tab i l ize r s co mp r ise th e 5 ' - u n t r an s la ted r eg io n

(UTR) of cer tain s tab le bacter ial and phage messages ,

such as the

ompA

tran scr ip t of

E. coli,

t h e

ermC

and

ermA trans cr ip ts of Bacillus subtilis an d Staphylococcus

aureus,

and the gene 32 mes sage of phage T4, as we ll as

a 5 ' segment of the phage \ p ^ t r an scr ip t (Yamamo to an d

Im am oto 1975; Gors ki et a l . 1985; Belasco et a l . 1986;

Bechhofer and Dubnau 1987; Sandler and Weisblum

1988).

Ap p aren t ly , th ese 5 ' e lemen ts a r e ab le to p ro tec t

mR NA f ro m a t tack b y an imp o r tan t ce l lu la r r ib o n u -

c lease th a t in i t i a tes mR NA d eg rad a t io n in v iv o ; th e

id en t i ty o f th i s k ey d eg rad a t iv e en zy me r emain s u n cer -

ta in . Mo s t o f th e k n o wn 5 ' mR NA s tab i l ize r s f u n c t io n

only under special condit ions . For example, the gene 32

5 ' UTR can in c r ease messag e l i f e t imes o n ly in T4 - in -

fected cel ls (Gorski et a l . 1985) , and s tab il izat ion of

m R N A b y t h e ermC a n d ermA 5 ' UT R s req u i r es rib o -

so me s ta l l in g in d u ced b y an an t ib io t ic th a t in h ib i t s

translat ion (Bechhofer and Dubnau 1987; Sandler and

We isb lu m 1 9 88 ). S imi la r ly , messag e s ta b i l iza t io n b y th e

5 ' por t ion of the X P t r an scr ip t i s mo s t p ro n o u n ced o n ly

GENES & DEVELOPMENT 6 :135-148 © 1992 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/92 $3.00

135


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Emory et al.

af te r p ro lo n g ed X in f ec t io n (Y amam o to an d Ima mo to

1975).

In contras t , the ompA 5 ' UT R can s tab i l ize mR NA in

£.

coli

under normal condit ions of rap id cel l g rowth

(Belasco et al. 1986; Em ory an d Belas co 1990). Th is RN A

segment is der ived f rom the

ompA

gene transcr i p t ,

wh ich en co d es a majo r E. coli o u t e r m e m b r a n e p r o t e i n

(OmpA). In cel ls growin g rapid ly at 30°C, the

ompA

m e s -

sage decays with a half-life of ~ 17 min, making it one of

t h e m o s t s t a b l e m R N A s i n E. coli (von Gabain et al.

1983). Th e l i f e t ime o f ompA m R N A is g ro wth ra te r eg-

u lated and fal ls by as much as a factor of 4 in s lowly

growing cells (Nilsson et a l . 1984) . Growth rate regula-

tion of ompA m R NA stabil i ty , l ike the longevity of the

ompA

trans cr ip t unde r condit io ns of rap id growth , is de-

te rmin ed b y th e 1 3 3 -n u c leo t id e ompA 5 ' UTR , wh ich

can confer both of these proper t ies onto o ther messages

to which i t is jo ined (Emory and Belasco 1990) .

As the l i fet imes of a var iety of lab ile messages can be

prolonged by fusion to the ompA 5' UT R (Belasco et al.

1986;

M. Ha nse n and J . Belasco , unpub l. ) , i t appears th at

m a n y

E. coli

mR NAs a r e d eg rad ed v ia a co mmo n p a th -

way ag a in s t wh ich th i s 5 ' R NA seg men t p ro v id es p ro -

tec t io n . M essag e s tab i l iza t io n b y th e

ompA

5 ' UT R is

not an indirect consequence of close r ibosome spacing ,

wh ich th eo re t ica l ly mig h t s te r ica l ly h in d er r ib o n u c lease

attack (Lundberg et a l . 1988; Emory and Belasco 1990;

M. Han sen and J. Belasco , unpu bl. ) . Ins tead , th is R NA

seg men t seems to ac t d i r ec t ly to p ro tec t mR NA f ro m

degradation by a r ibonuclease that responds to one or

mo re e lemen ts n ear th e mR NA 5 ' en d . B o th th e seco n d -

ary s tructure of the

ompA

5 ' UT R and i ts ab il i ty to func-

t io n as a g ro wth - r a te - r eg u la ted m R N A s tab i lize r a r e

highly conserved among enter ic bacter ia (Chen et a l .

1991). However , the identi ty of the specif ic ompA 5 '

UTR s t ru c tu r a l f ea tu r es th a t a r e imp o r tan t f o r mR NA

stabil izat ion has not been explored previously .

To e lu c id a te th e mech an ism o f mR NA s tab i l iza t io n

b y th e

E. coli ompA

5 ' UTR , we h av e d is sec ted th i s R NA

seg men t to id en t i fy th e s t ru c tu r a l co mp o n en ts th a t can

p ro tec t mR NA f ro m d eg rad a t io n in £ .

coli.

Ou r s tu d ies

reveal that the presence of a s tem-loop at or very near

th e ex t r eme 5 ' t e rmin u s o f th e ompA 5 ' UT R is essen tial

for i ts function as a potent mRNA stabil izer . Fur ther-

more, the half - l i fe of a normally lab ile E. coli messag e

can be pro longed by adding a s imple s tem-loop at i ts 5 '

en d . Th e 5 ' s tem- lo o p p ro tec t s ompA mR NA f rom d eg-

radation that appears to begin downstream of the 5 ' end .

By def in ing impo r ta nt aspec ts of the sub strat e specif ici ty

of the

E. coli

mR NA d eg rad a t io n mach in ery , th ese f in d -

ings provide key insights in to both the s tructural basis of

d i f f e r en t ia l messag e s tab i l i ty in b ac te r ia an d th e mech -

an ism o f mR NA d ecay .

Results

Two domains of the o m p A 5 ' UTR are important for

mRNA stabilization

We h av e sh o wn p rev io u s ly th a t th e E. coli ompA 5 ' UT R

comprises two imperfect s tem—loop s tructures (hpl and

hp2) and two s ingle-s tranded RNA segments (ss l and

ss2) (Fig. 1; Chen et al. 1991). The ompA 5 ' UT R was

d is sec ted to id en t i fy th e s t ru c tu r a l d o main s r esp o n s ib le

for i ts act iv i ty as an mRNA stabil izer in rap id ly growing

E. coli c ells . Var ious 5 ' UT R segm ent s we re delete d f rom

a p lasmid-borne copy of a pseudo-wild- type ompA gene

{ompA

+ 4]

th a t en co d es a t r an scr ip t v i r tu a l ly id en t ica l

in sequence and s tab il i ty to the wild- type

ompA

messag e

(Emory and Belasco 1990) . Th ese dele ted se gm ents cor-

responded to one or more RNA structural domains of the

ompA 5 ' UTR u p s t r ea m of th e r ib o so me-b in d in g s i te

(Fig. 1). Plasm ids enco ding the resu l t ing m ut an t

ompA

messag es were in t ro d u ced in to E. coli s train C600S, and

to tal cel lu lar RNA was iso lated f rom rapid ly growing

cu l tu r es a t t ime in te rv a ls a f te r t r an scr ip t io n in h ib i t io n

wi th r i f amp ic in . Deg rad a t io n o f each mu tan t messag e

and of the endogenous wild- type E. coli ompA messag e

was mo n i to r ed s imu l tan eo u s ly b y S I an a ly s i s o f th ese

RN A sa mpl es (Table 1). Th e paral lel analy sis of the de-

cay of wild-type ompA mR NA p ro v id ed an in te rn a l s tan -

dard that faci l i ta ted in terpretat ion of s tab il i ty d if fer -

en ces amo n g th e v ar io u s mu tan t t r an scr ip t s .

Two d is t in c t s t r u c tu r a l d o main s ap p ear to co n t r ib u te

to mR NA s tab i l iza t io n b y th e ompA 5 ' UT R. On e is the

5 ' - te rmin a l s tem - lo o p s t ru c tu r e (h p l ; F ig . I ) , wh o se p r e -

cise deletion reduces the half-life of ompA m R N A b y a

factor of 3 to just 5.7 ± 0.4 min {ompAA64) Fig. 2, left).

20

G U

A A

A U

G C

C G

C G

G U

V A G

: A u

G U

G U

C G

U A

C G

G C

U A

1 ^

A

1 u

G C

G C

G C

G C

A

A

G

U

u

A

-

A C -6 0

C G

C G

^40

U

G

C

U

C

U

A

U

U

G

U

G

C

G UGAAGGAUUUAAC

u

G

G

A

G

A

U

A

U

U

c

A -100

U

G

120

1

GCGUAUUUUGGAUGAUAACGAGGCGCAAAAAAUG

•

hpl ss1 hp2

ss2

Figure 1. Secondary stru cture of the

ompA

5' UTR. Brackets

delineate the boundaries of the four secondary-structure do-

mains w ithin this RNA segment (Chen et al. 1991): hpl (nucle-

otides 1-63), ssl (64-74), hp2 (75-103), and ss2 (104-133). The

Shine-Dalgamo element and translation initiation codon are

underlined. Precise

5'-end

mapping by primer extension shows

that roughly equal numbers of E. coli orapA transcripts begin

with the indicated 5'-terminal nucleotide or with a cytosine

residue 1 nucleotide upstream (data not shown).

136 GENES & DEVELOPMENT



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mRNA stabilization by a 5' stem- loop

Th e other is a single-stranded R NA segme nt (ss2; Fig. 1),

the 3' portion of which contains the ribosome-binding

site. The stabiUty of

ompA

mRN A is halved by deleting

from ss2 an 11-nucleotide RNA segment (5'-ss2) located

a few nucleotides upstream of the

ompA

Shine-Dalgamo

sequence

{ompAA104-114)

Fig.

2,

middle). Larger dele-

tions that remove either of these two elements (hpl or

5'-ss2) also destabilize the

ompA

message

{ompAA104,

ompAAllS)

Table 1). In contrast, deletio n of the inte rnal

hairpin (hp2) within the 5' UTR and of the single-

stranded segment (ssl) between hp l and hp2

[ompAA65-

104, onipAA74-103)

has little or no effect on the degra-

dation rate of the

ompA

transcript (Fig. 2, middle; Table

1).

All of these deletions leave the

ompA

ribosome-bind-

ing site intact, and none impair translation of

ompA

mRNA (Emory and Belasco 1990; Emory 1991).

Stabilization by the 5'-terminal stem—loop

is independent of its sequence

The 5' UTRs of

ompA

mRNA from

Seiratia maicescens

an d

Enterobacter aerogenes

function as effective mRNA

stabilizers in

E. coli

despite extensive sequence differ-

ences in both hpl and hp2 (Chen et al. 1991). To assess

the importance of the sequence of hpl to

ompA

mRNA

stability, the degradation of variant

E. coli ompA

mes-

sages with altered 5'-terminal hairpins was examined

(Fig. 3; Table 2). Tru nca tion of hp l by deleting 44 nucle-

otides from its top reduces th e ^df-life of the

ompA

mes-

sage by

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Emory et al.

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Table 1. Half-lives of mutant ompA transcripts lacking 5'

UTR segments

ompA

allele

Mutant

5' UTR half-life

structure'' (min)

Wild-type

half-life

(min)''

mRNA stabilization by a 5' stem-loop

ompA

+

4"

(pseudo-wild- type)

ompAl ^64^

(Ahpl)

ompAM04-114'-'^

(A5'-ss2)

ompAM4-103'^

(Ahp2)

ompA^65-104^

(Assl, hp2)

ompAM04^'^

(Ahpl , ssl , hp2)

ompAMlS^'^

(Ahpl , ssl , hp2, 5 '-

-ss2)

XL

\

\l

If

f

—

.._-

19 ±

1'^

SJ ±0.7

9.5 ± 0.8

14 ± 1

1 7 ± 1

4.4 ± 0.2

3.6 ± 0.6'^

N D

16 ± 2

18 ± 1

14 ± 1

19 ± 1

1 7 ± 3

1 9 ± 4

' 'The expected secondary structure of each mutant 5' UTR is

represented diagrammatically (for a detailed secondary struc-

ture of the wild-type 5' UTR, see Fig. 1). All were transcribed

from the hla promoter, whose transcription initiation site was

mapped precisely by primer extension (data not shown). (Solid

line) Retained 5' UTR segment; (dotted or absent line) deleted 5'

UTR segment; (solid rectangle) Shine-Dalgarno element.

''The half-life of the endogenous wild-type

ompA

message was

measured simultaneously in the same strain as each mutant

transcript. (ND) Not determined.

'=The ompA + 4, ompAM4-103, and ompAM04-114 5' UTRs

differ at the 5' end from that of wild-type

ompA

mRNA in that

the 5'-terminal G has been replaced by the sequence GAUCA.

The last 3 nucleotides of this substituted pentanucleotide

(UCA) are expected to augment hp 1 by 2 bp by pairing with the

last nucleotide of hpl (U) and the first 2 nucleotides of ssl (GA)

(see Table 2).

''Emory and Belasco (1990).

'^In addition to deletion of the number of 5'-terminal nucle-

otides indicated by the ompA allele name, 4 nucleotides

(GAUC) have been added at th e 5' end of ompA/: i64 mRNA, and

6 nucleotides (GAUCAG) have been added at the 5' end of

ompAM04

and

ompAMlS

mRNA.

'Segment

5'-ss2

comprises

ompA

nucleotides 104—114.

®In ompAA65-104 mRNA, the G-U pair normally at the bottom

of hpl has been changed to an A-U pair, and hpl has been

augmented at its base by 4 additional nucleotide pairs (GAUC-

hpl-GAUC). Furthermore, the deleted RNA segment (ssl-hp2)

has been replaced by 2 nucleotides (AG).

Owin g to th e p r esen ce o f th e w i ld - ty p e ompA t r an -

scr ip t in s train C600S, i t was unclear in i t ia l ly whether

th e r ap id d i sap p earan ce o f th e ex ten d ed ompA messag es

r ep resen ted mR NA d eg rad a t io n o r mere r emo v a l o f th e

single-s tranded por t ion of the 5 ' ex tension to generate a

s tab le p ro cess in g p ro d u c t r esemb l in g w i ld - ty p e ompA

U A

C G

G C

U A

G C

G C

G C

G C

A C

C G

C G

A U

C G

G A U A A G G A U .

ompA+4

G C

G C

G C

A C

C G

C G

A U

C G

G A U A A G G A U .

ompAA9-52

G CA AGG AU. . .

G A U A A G G A U .

ompAA64 ompAA29

Figure 3. Variant 5' stem-loop structures . For each ompA

message with an altered 5' hairpin, the sequence and expected

secondary structure of a 5'-terminal RNA segment tha t extends

into ssl are shown. In every case, the remainder of the mRNA

sequence is identical to that of the wild-type ompA transcript.

Adenosine, cytosine, and uridine residues susceptible to signif-

icant methylation by DMS in £.

coli

are indicated for the 5'-

terminal segment of

ompAA29

mRNA. (#) Heavy methylation;

(•) moderate methylation; (A) weak methylation.

mR NA . If s tab le , su ch a p ro cess in g p ro d u c t wo u l d b e

ex p ec ted to accu mu la te in th e ce l l to a s tead y - s ta te co n -

cen t r a t io n h ig h er th a n th a t o f i t s mo re lab i le p r ecu r so r s ,

the fu l l- length ompA t r an scr ip t s w i th 5 ' ex ten s i o n s . To

d is t in g u ish th ese p o ss ib i l i t i e s , R NA was i so la ted f ro m

an isogenic host s train (SE600) that bore a chromosomal

delet ion of the

ompA

g en e an d ca r r ied p lasm id

pOMPA-h 16a or pOMPA-l- 16b . SI and pr imer-extension

an a ly s i s of th ese s tead y - s ta te R NA sam p les sh o we d l i t t l e

or no detectable ompA + 16a

OT

ompA + 16b m R N A p r o -

cess in g p ro d u c t th a t was s imi la r in len g th to th e w i ld -

ty p e ompA mes sage (Fig . 5 , cont ro l lane s; Fig. 6) . The re-

fore,

addit io n of a shor t , s ingle-s t randed R NA se gm ent t o

Table 2. Half-lives of mutant ompA transcripts with variant

5' hairpins

ompA

al lele

ompA + 4

ompAA9-52

ompAA64*

ompAA29

m u t a n t

m e ssa g e

1 9 ± 2 ' '

13 ± 1

12 ± 1

15 ± 2

Half-life (min

1

wi ld- type

message^

N D

1 8 ± 2

1 8 ± 3

1 7 ± 2

^The half-life of the endogenous wild-type ompA message in

each strain was measured simultaneously. (ND) Not deter-

mined.

''Emory and Belasco (1990).

GENES & DEVELOPMENT 139



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Emory

et

al.

Table 3.

Mutant

ompA

transcripts with or without

destahihzing 5' extensions

ompA al lele

ompA

ompA

+

4

ompA + 12a

ompA + 16a

ompA + 16b

ompA + 12a*

ompA + 16b*

ompAM3a

ompAA73b

ompAA104»

5 '

U T R

st ructure^

(

L

H

ii

2

1

*

1

M u t a n t

half-life

(min)

19

±2

3. 2

± 1.1

5.8

± 0.3

6. 2

+ 0.8

13

± 1

13

+ 1

6. 0

± 0.3

13

+ 1

24

± 3

Wild- type

half-life

(min) ' '

17

± 2

N D

15

± 3

16

± 2

12

+ 1

17

± 1

15

+ 1

14

± 1

15

+ 1

19

+ 3

^The secondary structure

of

each muta nt 5' UTR is represented

diagrammatically. Only 5'-terminal single-stranded extensions

>2 nucleotides in length are show n. (Solid rectangle) Shine-

Dalgamo element;

[1] ompA

hpl; (2)

ompA

hp2; (*) synthetic

hp*.

''The half-life of the endogenous wild-type ompA message in

each strain was measured simultaneously. (ND) Not determined.

•^Emory and Belasco (1990).

t h e

5' end of

ompA

m R N A d e s t ab i l iz e s

th e

en t i r e t r an -

script.

Five unpaired nucleotides at the 5' end can destabilize

o m p A

mRNA

A n

ompA

d e l e t i o n m u t a n t

[ompAAJSa]

lack in g h p l and

s s l an d b eg in n in g in s tead w i th h p 2 - s s2 p r eced ed b y ju s t

5 n u c leo t id es (GAUC A) d ecay s w i th a sho rt half-life

(6.0

±

0 .3 min ) co m p ared wi th w i ld - ty p e ompA m R N A

(14 ± 1 min) (Table 3; Fig. 7). As a 5 ' h a i rp in of any k ind

a n d

ss 2

ap p ear

to be the

only features

of the

ompA

5 '

U T R t h a t are n ecessa ry for m R N A l o n ge v i ty , the shor t

l i f e t ime

of

ompAA73a mR NA su g g es ts th a t

a

5 ' - te rmi-

n a l s in g le - s t r an d ed seg men t o n ly 5 n u c l e o t i d e s in len g th

m a y

be

e n o u g h

to

acce le r a te ompA m R N A d eg rad a t io n .

Th is h y p o th es i s was co n f i rmed by m e a s u r i n g th e decay

ra te of a m e s s a g e

{ompAAJSb]

t h a t is id en t ica l to

ompAA73a ex cep t th a t it h as o n ly 1 nuc leo tide (A) up-

s t r e a m of hp 2- ss 2 (Table 3). As expec ted , t he half - l i fe of

ompAA73b m R N A (1 3 ± 1 min ) p ro v ed to be a b o u t as

long as t h a t of the wi ld - ty p e

ompA

tran scr ip t (15 ± 1

min) (Fig.

7).

Th e se f in d in g s in d ica te th a t

as few as 5

u n p a i r ed b ases at the 5 ' end are suff icien t to target

ompA

m R N A

for

rap id d eg rad a t io n

in

E. coli.

Addition

of a

5'-terminal stem-loop

can

prolong

the lifetime

of a

normally short-lived mRNA

A s a f ina l d emo n s t r a t io n th a t a 5 ' s t e m - l o o p and the

s in g le - s t r an d ed R NA seg men t en co mp ass in g th e ompA

r ib o so me-b in d in g s i te are suff icien t for th e full efficacy

of th e

amp A

mR NA s tab i l ize r , a m i n i m a l 5 ' U T R c o m -

pr is ing only a sy n th e t ic te rm in a l h a i rp in (hp *) and the

ompA

s s2 seg me n t was ev a lu a ted . As sh o w n

in

Figure

7,

a n ompA t r an scr ip t b ear in g th i s mi n im al

5' UT R

{ompAA104*) Tab le 3 )

is as

s tab le

as

th e w i ld - ty p e mes -

sage .

It is also f ive t im es m ore s ta b le tha n a s imi la r m es -

00 1 -

10

N ^ ^ ~ ^

•

o'\

ô

A o m p A + 1 2 a »

• o m p A + 1 6 a

O o m p A + 1 2 a

•_

•

^

•"

5 CAGACUUUACAUC

10 20

Time (min)

loo t

10

N

•

A o m p A + 1 6 b »

• o m p A + 1 6 b

,

\

s

• N

\

^

5 ' g c a G A U C U A U A C U A U A A C C . . .

^_*

0 15 30 45

Time (min)

Figure

4.

Decay

of

mutant ompA transcripts with

5'

exten-

sions.

After transcription inhibition, total cellular RNA was

isolated periodically from

E.

coli strain C600S containing

pOMPA-l-12a, pOMPA-Hl6a, or pOMPA -l-12a»

[top]

or con-

taining pOMPA-l- 16b or pOMPA-l- 16b»

[bottom].

The mutant

transcripts and the endogenous wild-type ompA message were

detected by SI analysis, and mRNA concentration was plotted

semilogarithmically as a function of time. Beside the graphs are

drawn the 5' extensions of ompA

+ 12a* [top]

and ompA -\-16b*

[bottom]

mRNA. The 5' extensions of

ompA + 12a

and

ompA-\-16b mRNA

are

related

to

those

of

ompA-\-12a*

and

ompA + 16b* mRNA, respectively, but include only those nu-

cleotides shown in uppercase letters. The 5' extension of

ompA

+ 16a

mRNA is identical to that of ompA

+ 12a

except for

4 additional nucleotides (GAUC) at th e 5 ' end. Underlined nu-

cleotides

at

the 3' end

of

each extension are expected to base-

pair with the first 1-2 nucleotides of 5' UTR segment ssl . Tran-

scription initiation sites

for

these messages were mapped pre-

cisely by primer extension (data not shown). The measured half-

lives were 3.2 ± 1.1 min for ompA-\-12a mRNA (O) and 15 ± 3

m in for wild-type ompA m RNA

[top],

5.8 ± 0.3 mi n for

ompA + 16a

mRNA ( ) and 16 ± 2 min for wild-type

ompA

mRNA [top], 13

± 1 min for

ompA-\-12a* mRNA

(A) and

17

±

1 min for wild-type ompA mRNA [top], 6.2

±

0.8 min for

ompA

+

16b mRNA ( ) and 12 ± 1 m in for wild-type ompA

mRNA [bottom], and 13 ± 1 min for ompA

+

16b* mRNA (A)

and 15 ± 1 min for wild-type

ompA

mRNA

[bottom].



http://file//-/-16b*http://www.cshlpress.com/http://www.cshlpress.com/http://genesdev.cshlp.org/http://genesdev.cshlp.org/http://www.cshlpress.com/http://genesdev.cshlp.org/http://file//-/-16b*

8/16/2019 Emory 1992 E.coli

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mRNA stabilization by a 5' stem -loop

sage

{ompAA104)

with a 5' UTR consisting only of the

ss2 segment (Table 1; Fig. 7). Thi s finding confirms tha t

a simple 5'-terminal hairpin and the

ompA

ss2 segment

together are as effective as the com plete

ompA

5' UTR at

stabilizing mRNA. Moreover, when the same synthetic

stem-loop (hp*) was introduced at the 5' terminus of

bla

mRNA, which encodes p-lactamase, the resulting

bla202

messages were found to decay with a half-life of

6.8 ± 0.4 min (Fig. 7), about twice the lifetime of wild-

type

bla

mRNA (3.7 ± 0.3 min) (Fig. 7; von Gabain et al.

1983).

Thus, the ability of

bla

mRNA to resist degrada-

tion in

E. coli

can be enhanced simply by adding a st em -

loop at its 5' end.

Degradation in the absence of a 5'-terminal stem-loop

appears to begin downstream of the

ompA 5'

end

In principle, accelerated degradation of

ompA

mRNA

lacking a 5'-terminal hairpin migh t be initiated either by

a 5' exonuclease (defined here as a ribonuclease that re-

moves single nucleotides sequentially from the RNA 5'

terminus) or by a 5'-end-dependent endonuclease that

cuts internally but is sensitive to the presence or absence

of secondary structure at the 5' terminus of mRNA. Of

these two possibilities, degradation by a single-strand-

specific 5' exonuclease seems less likely because no 5'

exoribonuclease has ever been detected in £.

coli

and

because destabilizing single-stranded extensions added

upstream of

ompA

h pl are not preferentially removed

from the 5' end of

ompA + 16a

a nd

ompA + 16b

mRNA

to generate wild-type-like

ompA

mRNA as a readily de-

tectable processing product. As 5' exoribonucleases, by

definition, digest RNA from the 5' end, the plausibility

of 5'-exonucleolytic initiation of mRNA decay can be

tested further by determining whether degradation of the

5' mRNA segment is faster or slower than decay of the

rest of the message.

Degradation of three different

ompA

messages with-

out a 5'-terminal stem-loop

{ompAA64, ompA + 16a,

an d

ompA

+

16b

mRNA) was monitored by SI analysis

with a mixture of two DNA probes, one complementary

to the 5'-terminal segment of these transcripts (the 5'

UTR and codons 1-37) and the other complementary to

an internal

ompA

segment spanning codons 67-295. By

combining these two probes in each SI protection assay,

differences in the relative decay rates of the two RNA

segments could be measured w ith considerable accuracy.

As observed previously for wild-type

ompA

mRNA (von

Gabain et al. 1983), the internal segment of all three of

these m uta nt messages was found to decay more rapidly

than the corresponding 5'-terminal segment (Fig. 8). To

demonstrate that the differential lifetimes measured for

the 5'-terminal and internal

ompA

mRNA segments are

independent of the relative lengths of the two DNA

probes used simultaneously for SI protection, the mea-

surements were repeated with the same 5'-terminal

probe and a mu ch shorter internal probe com plementary

to

ompA

codons 249 -295 . Regardless of the length of

either the internal probe or the RNA segment with

which it hybridized, the same segmental difference in

stability was observed for each of the three mutant

ompA

messages (Fig. 8). In every one of these six inde-

pendent experiments, the relative concentration of the

internal mRNA segment declined steadily to a level be-

tween one-third and one-half that of the 5'-terminal seg-

ment within 30 min after transcription inhibition.

Thus,

it appears for three different labile

ompA

tran-

scripts lacking a 5'-terminal stem-loop that degradation

of an internal RNA segment precedes decay of the 5'

UTR. This conclusion is consistent with our finding, for

a number of mutant

ompA

messages, that there is no

correlation in rapidly growing cells between m RN A half-

life and the relative steady-state concentration of the

major products of endonucleolytic cleavage within the 5'

UTR (data not shown; Lundberg et al. 1990); only when

ompA

mRNA decay accelerates in slowly growing cells

does its rate appear to be controlled by 5' UTR cleavage

(Melefors and von Gabain 1988). Together, our data sug-

gest that under conditions of rapid bacterial growth, the

function of the 5'-terminal

ompA

hairpin is to protect

the message from degradation by a ribonuclease that ini-

tiates decay downstream of the 5' end.

Discussion

A key to understanding the molecular basis for differen-

tial mRNA stability in bacteria is to identify the struc-

tural features of stable messages that are responsible for

their unusual longevity in vivo. The studies reported

here show th at a 5'-termin al stem—loop is bot h crucial to

the function of the

ompA

5' UTR as a potent mRNA

stabilizer and sufficient to prolong the lifetime of a nor-

mally labile

E. coli

message. Remarkably, the 5'-termi-

nal stem-loop shields

ompA

mRNA from attack by a

cellular ribonuclease that appears to initiate degradation

far from the 5' end.

Our data also indicate that the

ompA

mRNA stabilizer

is bipartite, with a second, distinct domain of functional

importance. Both the 5' stem -loop and a single-stranded

RNA segment in the vicinity of the

ompA

ribosome-

binding site and its flanking sequences contribute to th e

remarkable longevity of the

ompA

transcript, and to-

gether they are sufficient for full activity of the

ompA

mRN A stabilizer. Alone, each of these elem ents can sta-

bilize mRNA to a lesser extent. Thus,

ompA

transcripts

that have both stabilizing elements (wild-type,

ompAA65-104]

are more stable than

ompA

messages

lacking either the 5' hairpin or

5'-ss2

[ompAA64,

ompAA104, ompAA104-114]

which, in turn, are more

stable than a message that lacks both of these RNA seg-

ments

[ompAAllS].

Sim ilarly, the half-life of

bla

mRNA , wh ich is increased twofold simply by adding a 5'

s tem-loop

[bla202],

increases about fivefold when its 5'

UTR is replaced with the entire

ompA

5' UTR (Belasco

et al. 1986; Emory and Belasco 1990).

The longevity of

ompAA104*

mRNA, in which hpl,

ss l ,

and hp2 are replaced by a synthetic stem -loo p, ru les

out any contributio n to

ompA

mRNA stability from pos-




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Emory et al .

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tnRNA stabilization by a 5' stem-loop

sib le s i tes of t ranslat ion in i t ia t ion upstream of ss2 .

These s i tes , which were identif ied on the basis of se-

quence analysis (Movva et a l . 1980) , probably are inac-

cess ib le to r ib o so me s d u e to o cc lu s io n b y in t r a mo le cu la r

base-pairing (Chen et al. 1991).

Stabilization of mRNA by a

5 '

stem-loop

Th e p resen ce o f a 5 ' - t e rmin a l s tem - lo o p can p ro lo n g th e

lifet ime of ompA m RN A by as m uc h as a factor of 5 . The

lo ca t io n of th i s s tem - lo o p a t o r v e ry n ear th e 5 ' t e rm in u s

is crucial to i ts s tab il izing ef fect , whereas the sequence

of th is hairp in and i ts posi t ion relat ive to the r ibosome-

binding s i te appear to be of l i t t le consequence to message

s tab i l i ty . Up to two u n p a i r ed n u c leo t id es u p s t r eam o f

the 5 ' hairpin (e.g. , ompA+4, ompAA29] are to lerat ed

wi th o u t an y r ed u c t io n in mR NA s tab i l i ty , b u t th e ad d i -

t io n o f 1 0 -1 5 u n p a i r ed n u c leo t id es o f r an d o m seq u en ce

to the

ompA

5 ' end

{ompA + 12a, ompA + 16a,

ompA + 16b] is as destab il izing as delet ion of the 5 ' hair -

p in . Moreover , the shor t l i fet ime of

ompAA73a

m R N A

v er su s ompAAlSb m R N A in d ica tes th a t 5 u n p a i r ed

bases at the 5 ' end are suff icien t to accelerate degrada-

tion of ompA mR NA , as th ese two messag es a re id en t i -

cal in sequence except for the number of nucleotides that

p r eced e h p 2 - s s2 . Th ere fo re , th e min imu m n u mb er o f 5 ' -

t e rmin a l u n p a i r ed n u c leo t id es th a t can d es tab i l ize ompA

m R N A i n E. coli is no more than f ive and may be as few

as three. The destab il izing ef fect of unpaired bases at the

ompA

5 ' t e rmi n u s d o es n o t ap p ear to b e seq u en ce sp e-

cific.

These f indings explain our previous observation that

the 5 ' UTRs of the

S. maicescens

an d

E. aerogenes ompA

t rans cr ip ts function as very ef fective mR N A stabil izers

in

E. coli

(Chen et a l . 1991) . Alt hou gh thes e

ompA

5 '

UTRs are quite s imilar in secondary s tructure to that of

E. coli and the sequence of ss l and ss2 is h ighly con-

served (80%), there is ex tensive sequence d ivergence in

hpl and hp2 (Chen et al. 1991). The efficacy of the Ser-

337 —

296 —

M

-

MM

m^

Figure 6. 5'-End m apping of ompA + 16a and ompA + 16b

mRNA at steady state. Total cellular RNA was isolated from E.

coli

strain C600S or from strain SE600 containing either

pOMPA-l-16a or pOMPA+16b. The wild-type and mutant

ompA transcripts were detected by SI analysis with 5'-end-la-

beled DNA probes complementary to the first 0.32-0.34 kb of

each message. Calibration is in nucleotid es. A set of m olecular

size standards (lane M) was generated as in Fig. 2.

ratia an d Enterobactei ompA 5' U T R s i n E. coli, d esp i te

n u m ero u s h p l an d h p 2 seq u en ce d i f fe r ences , i s n o w u n -

ders tandable in l igh t of our present f inding that v ir tually

any 5 ' - terminal hairp in fo l lowed by ss2 is suf f icien t to

p ro tec t ompA mR NA f rom rap id d eg rad a t io n .

As th e seq u en ce o f th e 5 ' s tem- lo o p i s r e la t iv e ly u n -

imp o r tan t to messag e s tab i l i ty an d i t s s tab i l iz in g in f lu -

ence is negated by the presence upstream of several un-

paired nucleotides , i t ev idently is the absence of a s ig-

n i f ican t s in g le - s t r an d ed R NA seg men t a t th e 5 ' t e rmin u s

ra th er th an th e p r esen ce o f ompA h p l t h a t m a k e s m e s -

sages bear ing the ompA 5 ' UT R res i s tan t to d eg rad a t io n .

We n o te th a t s ev era l o th er lo n g - l iv ed p ro k ary o t ic mes -

sages in their mature form (e.g . , E. coli papA m R N A , T 4

g en e 3 2 mR NA, an d Rhodobacter capsulatus pufBA

mR N A) h av e th e p o ten t ia l to fo rm a h a i rp in s t ru c tu r e

with in 1-4 nucleotides of the 5 ' end (Fig . 9) , (Youvan et

al.

1984; Belasco et al. 1985; Gorski et al. 1985; Baga et

al.

1988; Mc Phe ete rs et a l . 1988) . Tog ethe r wit h our

present f indings, th is observation suggests that a 5 '

Figure 5. Me thyla tion of

ompA

transcripts bearing 5' extensions.

[Top]

Total cellular RNA was isolated from £.

coli

strain SE600

containing pOMPA+16a

[left]

or pOMPA-t-16b

(right)

after treating each culture w ith DMS (DMS/in vivo). Alternatively, total

cellular RNA was purified from the sam e cultures with out prior DMS treatmen t and then a lkylated in vitro with either DM S (DMS/in

vitro) or CMCT. Sites of alkylation within the 5' UTR of ompA + 16a and ompA + 16 b mRNA were mapped by primer extension with

AMV reverse transcriptase, using complem entary oligodeoxynucleotides (AGCGA AACCAG CCAGTGC CACTGC or GATAACACG-

GTTAAATCCTTCAC) that annealed to mRNA sequences either 22-45 nucleotides downstream or 50-72 nucleotides upstream of

the translation initiation codon of

onipA + 16a

and

ompA + 16b.

Gel electrophoresis was performed in parallel with the products of

primer extension on unmethylated RNA templates in the presence (lanes

U, G, C, A]

or absence (control lane) of dideoxynucleoside

triphosphates. The sequencing lanes are labeled to indicate the sequence of the RNA, not the c omplem entary D NA. Blockage of prime r

extension by an alkylated RNA base results in a complementary DN A fragment t hat is 1 nucleotide shorter than that arising from

incorporation of a dideoxynucleotide opposite the same RNA base. Calibration is in nucleotides from the mRNA 5' end. (Bottom)

Summary of alkylation data for the 5' UTRs of ompA

+

lSa (left) and onipA

+

16b (right) mRN A. Adenosine, cytosine, and u ridine

residues susceptible to significant me thylation by DMS in

E. coli

are indicated. (•] Heavy methyla tion; (•) m oderate methy lation; (A)

weak me thylation . Also labeled are uridine and guanosine residues that are significantly alkylated by CMCT in vitro. (•) Heavy-to-

moderate alkylation; ( 0) weak alkylation. Com parisons of susceptibility to alkylation are meaningful only among nucleotides wit hin

the same message and at a similar distance from the annealed primer. Arrows identify the principal sites of termination by reverse

transcriptase on an unalkylated template; the structural significance of primer extension products ending at these sites on alkylated

RNA is often difficult to assess. Two of these termination sites on unalkylated RNA (at nucleotides 84 and 123) correspond to known

RNase K cleavage sites within the

ompA

5' UTR (Lundberg et al. 1990); whether or not the others also correspond to RNA 5' ends is

uncertain. N o differences w ere observed between the alkylation patterns of the ompA + 12a (data not shown) and ompA + 16a5' UTRs.




8/16/2019 Emory 1992 E.coli

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Emory et al.

Figure 7. Degrad ation of a variant ompA

transcript bearing a minimal 5' stabilizer

preceded by 0, 1, or 5 nucleotides and of

bla mRNA with an added 5'-terminal

stem-loop. After transcription inhibition,

total cellular RNA was isolated periodi-

cally from E. coli strain C600S containing

pOMPAA 73a (top left), pOMPAA 73b (bot-

tom

left),

pOMPAA304*

(top

hght),

pBLA200

{bottom hght),

or pBLA202

{bot-

tom hght).

Transcripts of the

ompA,

ompAA73a, ompAA73b, ompAA104*,

bla200, an d bla202 genes were detected by

SI analysis, and mRNA concentration was

plotted semilogarithmically as a function

of time. Th e mea sured half-lives we re

6.0

±

0.3

min for ompAA73a

mRNA

(A)

and 14 ± 1 min for wild-type ompA

mRNA (• ) {top left), 13 ± 1 min for

ompAAJSb mRNA (A) and 15 ± 1 min for

wild-type ompA mRNA ( ) (bottom

left),

24 ± 3 min for

ompAA104*

mRNA (A)

and 19 ± 3 min for wild-type ompA

mRNA

(• )

{top hght),

3.7 ±

0.3

min for

bla200 mRNA (wild-type) (•) and 18 ± 2

min for wild-type ompA mRNA {bottom

hght), and 6.8 ± 0.4 min for

bla202

mRNA

(A) and 17 ± 2 m in for wild-type

ompA

mRNA {bottom hght). For comparison, de-

cay data for ompAA104 mRNA (•) are also

shown {top hght; Table 1).

100

10

^r

A ompA A73o

• ompA

100

g 10

A ompA A l 04-*

• ompA A104

• ompA

0 15 30

Time min)

15

30 45

Time (min)

10011

10

• b la202

• b la200

2 0 40

Time min)

10

Time min)

20

S t e m - l o o p m a y be of g en era l imp o r tan ce as a m e a n s b y

wh ich p ro k ary o t ic o rg an isms

can

se lec t iv e ly en h an c e

th e s tab i l i ty of m R N A .

Mechanism

of

mRNA degradation

in E.

coli

Th e ro u g h ly ad d i t iv e co n t r ib u t io n s to m R N A s tab i l i ty of

two d is t in c t s t r u c tu r a l d o main s of the ompA 5' UTR

suggest that the degradation of messag es th a t can be sta-

b il ized

by

fusion

to

t h i s

5' UTR is

in i t i a ted e i th e r

by a

s in g le r ib o n u c lease wh o se r a te of a t tack i s d e te rm in ed b y

a c o m b i n a t i o n of 5' UT R s t ru c tu r a l f ea tu r es or by a pair

of r ibonucleases with d if fer ing specif ici t ies , bo th of

w h i c h m u s t be b lo ck ed to a c h i e v e p r o n o u n c e d m R N A

lo n g ev i ty . Un d o u b ted ly th e r e are u n s tab le t r an scr ip t s

wh o se lab i l i ty r esu l t s f ro m a t tack by a dif ferent r ibonu-

c lease th a t is n o t sen s i t iv e to s t ru c tu r a l f ea tu r es n e ar the

m R N A 5 ' en d ; ad d i t io n of a 5 ' - te rmi nal ha irp in or fusion

to th e en t i r e ompA 5' U T R is not ex p ec ted to stab il ize

su ch messag es . Nev er th e les s ,

the

l i f e t imes

of

m a n y

shor t- l ived E. coli R NAs ( in c lu d in g bla, lacZ, an d phoA

mRNA) are contro lled by the r ibonuclease(s) impeded by

s u b s t i t u t i n g

the ompA 5' UT R or

ad d in g

a

s i m p l e

5'-

t e rmin a l s tem- lo o p (B e lasco et al. 1986; M. Han sen an d

J. Belasco , unpu bl. ) ; and , in p r in c ip le , all long- l ived mes-

sag es mu s t h av e s t ru c tu r es , s eq u en ces , or bou nd factors

th a t sh ie ld th em f ro m a t tack by th i s en zy m e.

Our data therefore suggest that E. coli c o n t a i n s a r ibo-

n u c lease th a t p r e f e r s to a t tac k messag es b eg in n in g w i th

> 2 - 4 u n p a i r e d n u c l e o t i d e s . T h i s e n z y m e is no t a 3 ' ex-

o n u c lease , as m u l t i p l e l i n e s of ev id en ce in d ica te th a t bla

mR NA d eg rad a t io n , wh ich is s lo wed by ad d i t io n of a

5 ' - te rmin a l h a i rp in , b eg in s u p s t r eam of th e 3 ' end (von

G a b a i n et al. 1983; Belasco et al. 1986). Instead, the sen-

s i t iv i ty

of

th i s r ib o n u c lease

to

base-pair ing

at

t h e m R N A

5 '

te rmin u s in d ica tes th a t

it is

e i th e r

a 5 '

ex o n u c lease

or

a 5 '- en d -d ep en d en t en d o n u c lease th a t cu ts in te rn a l ly b u t

in te r ac ts , at leas t in i t ia l ly , w i th th e 5' en d of m R N A .

Th ere a r e a n u m b e r of r easo n s to d o u b t th a t the r ib o n u -

c lease o b s t ru c ted by the ompA 5' UT R is a 5' ex o n u -

c lease th a t r em o v es s in g le n u c leo t id es seq u en t ia l ly f rom

th e R NA 5 ' end. First, as sh o w n p rev io u s ly for the wild-

ty p e

ompA

trans cr ip t (von Gab ain et al. 1983), rapid deg-

r ad a t io n of onipAA64, ompA + 16a, and ompA + 16b

m R N A a p p e a r s to b e gi n d o w n s t r e a m of th e 5' U T R . In

addit ion , degradation of ompA tran scr i p ts wi th ei ther of

two s in g le - s t r an d ed 5' ex ten s io n s d o es no t g en era te

wi ld - ty p e ompA m R N A as a readily dete ctab le decay in-

te rmed ia te ; s ig n i f ican t accu mu la t io n of s u c h an in ter -

m e d i a t e m i g h t

be

ex p ec ted

if

th ese messag es were

de-

graded by a h y p o t h e t i c a l 5 ' e x o n u c l e a se t h a t is imp ed ed

w h e n it e n c o u n t e r s an R NA s tem - lo o p s t ru c tu r e . Th i rd ,

n o 5 ' ex o r ib o n u c lease h as ev er b een d e tec ted in E. coli.

Fin a l ly , in ac t iv a t io n of the

ams/rne

g en e p ro d u c t in E.

coli s tab i l izes man y R NAs ( in c lu d in g ompA mR N A) an d

in h ib i t s th e i r c leav ag e in v iv o by R N a s e E or R N a s e K,

t w o £. coli en d o n u c leases th a t ap p ear to be in te r r e la ted

a n d m ay ev en be ident ical (Apir ion 197 8; O no and Ku-

wan o 1 9 7 9 ; Lu n d b erg et al. 1 9 9 0 ; Mu d d et al. 1990a,b;

B ab i tzk e and Ku sh n er 1 9 9 1 ; L in -C h ao and C o h e n 1 9 9 1 ;

144 GENES DEVELOPMENT



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mRNA stabilization by a 5' stem-loop

100

10 -

ompA+16o

• 5'

• in ternal (6 7- 29 5)

100 '

>A onr)pA+1 6a

10

1

V ^

k N.

• 5'

A in te rna l (249 -295 ) .

10 20

Tlnne (min)

30 10 20

Tim*

(min)

30

100

10

onnpA+16b

• 5'

• in ternal (67 -29 5) f

IOOI 'A

10

ompA+16b

• 5'

• in ternal (2 49 -2 95 )

10 20

TlnDe (min)

30

10 20

Time (min)

30

l O O o

10

o m p M 6 4

• 5'

• in ternal (67 -29 5)

100

10

ompAA64

• in ternol (24 9- 29 5)

10 20

Time (min)

30

10 20 30

Time (min)

Figure 8. Segmental differences in stability with in mu ta nt

ompA transcripts lacking a 5'-terminal stem-loop. After tran-

scription inhibition, total cellular RNA was isolated periodi-

cally from E. coli strain SE600 containing pOMPA-l-16a,

pOMPA-l-16b, or pOMPAA64. The degradation of 5'-terminal

and internal segments of each mutant ompA transcript was

monitored simultaneously by SI analysis with a mixture of two

5'-end-labeled DNA probes, one complementary to the 5' UTR

plus codons 1-37 and the other comp lementary either to codons

67-295 or to codons 249-295.

[Top]

Semilogarithmic plots of

the decay of 5'-terminal (#) and internal (A) segments of

ompA + 16a [top], om pA + 16b {middle], and ompAA64 {bottom]

mRNA, as determined by Si analysis with tw o pairs of segment-

specific probes. In every case, decay of the internal segment is

faster than decay of the 5'-terminal segment.

{Below]

Map of the

5'

and internal

ompA

mRNA segments detected by SI analysis

with each probe. (Thin lines)

ompA

UTRs; (open rectangle)

ompA protein-coding region; (arrowhead) ompA 3' end; (thick

lines) ompA mRNA segments complementary to each of the

three probes.

in g en d o n u c leo ly t ic c leav ag e a t o n e o r mo re s i te s d o wn -

stream of the 5 ' end . The precise location of these s i tes is

cu r r en t ly u n d er in v es t ig a t io n .

I f th e en zy me th a t i s imp ed ed b y th e ompA 5 ' UT R is

an endonuclease ( i .e . , a r ibonuclease that cu ts in ter -

nal ly) ,

th en th e r a te o f mR NA c leav ag e b y th i s en d o n u -

clease is determined not only by the presence of RNA

sequences that can serve as cleavage s i tes but also by the

p resen ce o r ab sen ce o f s t r u c tu r es n ear th e mR NA 5 ' en d

that can contro l access to these s i tes . Indeed , the ab il i ty

o f a 5 ' - t e rmin a l s tem- lo o p to sh ie ld an en t i r e t r an scr ip t

f ro m d eg rad a t io n su g g es ts th a t th e p r imary d e te rmin an t

of mRNA half - l i fe may of ten be the accessib i l i ty of po-

ten t ia l c leav ag e s i tes r a th er th an th e i r n u mb er o r th e i r

in tr i ns ic susce ptib i l i ty to cleavage. Th is pro t ecti ve ef fect

o f a 5 ' s tem- lo o p su g g es ts a d eg rad a t io n mech an ism fo r

E. coli messages that can be s tab il ized by fusion to the

ompA

5 ' UTR , wh ere b y a r a te -d e te rm in in g , 5 ' -en d -d e-

p en d en t en d o n u c lease f i r s t b in d s in a s t r u c tu r e -d ep en -

d en t s tep to th e m R N A 5 ' en d o r to a p ro te in b o u n d th ere

o p

mRNA

249-295

67-295

intemal

u

A

U

U

G

G

U

C

U

A

G

JO

G

U

A

A

U

C

G

G

G

U

C

AUUUUA

G

G

G

U

G

G

G

C

A

C

C

U

C

D

GO

C

c

c

c

u

c

G

0

G

G

0

G

G

A

AAOGCG

0 A

G 0

C G

„ G

M ac k ie 1 9 9 1 ; M ele fo r s an d v o n Gab a in 1 9 9 1 ; N i l s so n

and Uh lin 1991; Tara sevic iene et a l . 1991) ; therefore,

ei ther or both of these endonucleases are candidates for

th e k ey en zy me( s ) wh o se ac t io n i s imp ed ed b y th e ompA

5 ' UTR. I t seems l ikely , therefore, that the ompA 5 ' UT R

p ro tec ts th e ompA mess ag e f ro m in i t ia l , ra te -d e te rmin -

papA

pufBA

gene 32

Figure 9. Stem-loop structures near the 5 ' end of stable

prokaryotic mRNAs. The sequence and likely secondary struc-

ture is shown for a 5'-terminal untranslated segment of E. coli

papA mRNA, R. capsulatus pufBA mRNA, and T4 gene 32

mRNA (McPheeters et al. 1988).

GENES

&,

DEVELOPMENT

145



8/16/2019 Emory 1992 E.coli

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Emory et al.

an d th en seek s o u t c leav ag e s i tes d o w n s t r ea m. Th e R NA

fragments thereby generated could be rap id ly degraded to

mo n o n u c leo t i d es b y fu r th er en d o n u c lease c leav ag e an d

ex o n u c lease d ig es t io n .

Th e in f lu en ce o f a 5 ' - t e rmin a l s t r u c tu r a l e lem en t o n

th e s tab i l i ty o f mR NA b ear s a s t r ik in g r esemb lan ce to

the select ive degradation of pro teins on the basis of their

amin o - te rmin a l amin o ac id r es id u e (Var sh av sk y e t a l .

1988). Moreo ver , the appa rent ef fect of an up stre am

s t ru c tu ra l e lemen t o n mR NA c leav ag e a t d o wn s t r eam

s i tes i s r em in isc en t o f ce l lu la r r eg u la to ry m ech an is ms

th a t co n t ro l t r an scr ip t io n an d t r an s la t io n r a tes v ia se -

q u en ce e lemen ts lo ca ted u p s t r eam o f in i t i a t io n s i tes f o r

R N A o r p ro te in sy n th es i s .

Mateiia ls and methods

Bacterial strains and plasmids

E. coli K-12 strains C600S and SE600 (Nilsson et al. 1987) are

streptomycin-resistant, supE44~ variants of strain C600. SE600

is identical to C600S except for a deletion of the chromosomal

ompA

gene (Emory and Belasco 1990).

Plasmid constructions were confirmed by restriction map-

ping and DNA sequencing. When necessary, protruding DNA

ends were made blunt prior to ligation by treatment with T4

DNA polymerase, the Klenow fragment of DNA polymerase I,

or mung bean nuclease. Oligonucleotide-directed mutagenesis

was performed as described previously (Kunkel 1985; Naka-

maye and Eckstein 1986) using purified oligodeoxynucleotides

synthesized on an Applied Biosystems 381A instrument.

Plasmid pOMPA-l-4 is a pBR322 derivative that encodes a

pseudo-wild-type ompA message transcribed from the bla pro-

moter (Emory and Belasco 1990); this plasmid has a

Bell

site at

th e

ompA + 4

transcription initiation site. Plasmid pOMPA-l-

4-1-3 was constructed by oligonucleotide-directed insertion of 3

bp (ATC) into pOMPA-l-4 to create a second Bell site at the

promoter-distal end of the segment encoding hpl (GTGAA-*

GTGATCAA). Deletion of the 0.07-kb Bell fragment of

pOMPA-f4-l-3 generated pOMPAA64. Plasmids pOMPAA29

and pOMPAA73a were c onstructed by deleting from pOMPA -I- 4 a

0.03-kb Bell (filled-in)-£coRV fragment or a 0.08-kb Bell (filled-

in)-Hphl (T4 DNA polymerase) fragment, respectively, thereby

reconstituting the Bell site. Plasmid pOMPAA73b was con-

structed by cleaving pOMPAA73a wi th Bell, treating the linear-

ized DNA with mung bean nuclease, and religating the result-

ing blunt ends. The 5' terminus of

ompAAlSb

mRNA mapped

to 1 nucleotide upstream of ompA hp2, and DNA sequencing of

pOMPAA73b revealed the unexpected loss of one additional

base pair upstream of the four that were planned. Plasmid

pOMPAA74-103 was constructed by oligonucleotide-directed

deletion of a 30-bp pOMPA -I- 4 fragment that encodes hp2 and

the preceding nucleotide; this mutation created a Sna^l at the

site of deletion. Plasmid pOMPAA104 was created by deleting

the 0.08-kb Bell-Snam fragm ent of pOMP AA74-103, after first

adapting the SndSi end with a Bell linker (CTGATCAG) and

cleaving the linker with

Bell.

Insertion of the 0.06-kb

Bell

frag-

ment of pOMPA -1-4-1-3 in its natural orientation into the

Bell

site of pOMPAA104 generated pOMPAA65-104. Plasmids

pOMPAA9-52 and pOMPAA104-114 were constructed by oligo-

nucleotide-directed deletion from pOMPA-f4 of either a 44-bp

fragment encoding th e top of hpl or an 11-bp fragment encoding

the first 11 nucleotides of ss2, respectively.

Plasmid pOMPA-l- 16a was created by cloning a 1.28-kb AccI

(filled-in)-PstI ompA fragment of pTUlOO (Bremer et al. 1980)

between the Bell (filled-in) an d Pstl sites of pJB322 (Belasco et al.

1986),

thereby reconstituting the BeR. s ite. Plasmid pOMPA-h 12a

was constructed by cleaving pOMPA-l- 16a with

Bell,

removing

the protruding ends with mung bean nuclease, and religating

the resulting blunt ends. Plasmid pOMPA-l-21b was con-

structed by oligonucleotide-directed insertion of 17 bp (GATC-

TATACTATAACCG) at a site immediately promoter-distal to

th e

Bell

recognition sequence of pOMPA-l-4, thereby creating a

Bglll

site adjacent to the

Bell

site (TGATCAGATCTATAC-

TATAACCG). Plasmid pOMPA-hl6b is nearly identical to

pOMPA-l-21b but lacks the 5-bp Bell-BglU fragment. Insertion

of a symmetrical DNA linker (GCCCACCGGCAGCTGCCG-

GTGGGC) into the Bell site (filled-in) of pOMPAA64,

pOM PA+ 16a, pOMPA

21b,

pOMPAA104, and pBLA200 (Em-

ory and Belasco 1990) created pOMPAA64», pOMPA-l-12a*,

pOMPA-l- 16b*, pOMPAA104*, and pBLA202, respe ctively.

Plasmid pOMPAA331 was constructed by deleting a 0.33-kb

Bell (filled \n]-Hpal fragment from pOMPA-l- 16a, thereby re-

constituting the Bell site. Plasmid pPBlOl was created by in-

serting a 0.39-kb

BamHl-Pstl

fragment of pOM PA+ 16a be-

tween the BflmHI and Pstl sites of pUC19.

Measurement of mRNA lifetimes

Bacterial culture at 30°C in supplemented LB medium, RNA

extraction, SI analysis, and calculation of mRNA half-lives

were performed as described previously (Emory and Belasco

1990).

All strains grew rapidly under these conditions, with dou-

bling times between 34 and 44 min. Q uantita tion of SI-protec-

tion data was accomplished either by autoradiography and den-

sitometry (Emory and Belasco 1990) or by gel scanning on a

Molecular Dynamics model 400 Phosphorlmager. Half-lives

were calculated by least-squares analysis of semilogarithmic

plots of mRNA concentration versus time. Half-life errors were

estimated from the standard deviation of the slope of each plot.

Three different 5'-end-labeled DNA fragments of pOMPA-l- 16a

were used as probes to monitor the decay of wild-type

ompA

mRNA and most variants

thereof.

SI analysis with any of these

probes (a 1.2-kb SgiII-£coRI DNA fragment, a 0.6-kb BstEII-

£coRI fragment, or a 0.6-kb //indlll-fcoRI fragment), which

have be en described prev iously (Emory and B elasco 1990), gives

the same half-life for the wild-type

ompA

transcript because all

three probes extend beyond the 3' boundary of the compara-

tively stable 5'-terminal segment of ompA mRNA. Decay of

ompA + 16 b and ompA + 16b* mRNA was monitored by SI

analysis with a 0.6-kb Hindlll-EeoRl fragment of a

pOMPA-l-

16b*

point mutan t, which was 5'-labeled at a Hindlll

site created by oligonucleotide-directed mutagenesis at a site

corresponding to ompA codon 94. Differential decay of seg-

ments within the ompA + 16a, ompA + 16b, an d ompAA64 mes-

sages was monitored by simultan eous SI analysis with two seg-

ment-specific probes labeled at the 5' end: a 0.46-kb Avall-

Hindlll

fragment of pOMPA-l-16a complementary to the 5'

UTR plus codons 1-37, and either a 0.86-kb BgiII-£coRI frag-

ment of pOMPAA331 complementary to codons 67-295 or a

0.16-kb BgiII-£coRI fragment of pPBlOl complementary to

codons 249-295. Comparative 5'-end-mapping of ompA,

ompA

+ 16a,

and ompA

+

16b mRNA was performed by SI anal-

ysis with a 0.5-kb

BstEU-EeoW.

fragment of eithe r pOMPA-H 16a

or pOMPA-l- 16b. Decay of blaZOO and bla202 mRNA was mon-

itored by SI analysis with a 5'-labeled

1.03-kb

Hinil-Hindlll

fragment of pBLA200.

Probing of RNA seeondaiy structure w ith DM S

Methyla tion of RNA by DMS in £.

eoli

and in vitro, alkylation

of RNA with CMCT in vitro, and mapping of reactive nucle-

146

GENES & DEVELOPMENT



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mRNA stabilization by a 5' stem -loop

ot ides were performed as descr ibed previously (Moazed et al .

1986;

Ch en e t a l . 1991) . Bacter i a for DMS and CM CT alkyla t ion

(SE600/pOMPA+12a, SE600/pOMPA+16a, SE600/pOMPA+16b,

or SE600/pOMPAA29) were grown and harves t ed under t he

sa m e c o n d i t i o n s a s w e r e e m p l o y e d fo r m e a su r e m e n t s of m R N A

stabi l i t y . Two 5 ' - end- l abeled DNA ol igonucleot ides (AGC-

G A A A C C A G C C A G T G C C A C T G C o r G A T A A C A C G G T T A -

A A T C C T T C A C ) w e r e u se d a s p ri m e r s t o m a p m e t h y l a t i o n s i t es

wi th in the 5 ' UTR of

ompA

m R N A v a r i a n t s . T h e se p r i m e r s

a n n e a l e d t o m R N A se q u e n c e s 2 2 - 4 5 n u c l e o t i d e s d o w n s t r e a m

or 50-72 nucleot ides upst r eam of t he ompA t r ans l a t ion in i t i a -

t i on codon, r espect ive ly , and they were extended a t 55°C wi th

AMV rever se t r ansc r ip t ase (Boehringer) .

A c k n o w l e d g m e n t s

We thank L.-H. Chen, I . Laird-Offr inga, and C. Well ington for

the i r he lpfu l com me nt s and M. Han sen for a p l asm id cons t ruc-

t i on . Thi s work was suppor t ed by U.S. Publ i c Heal th Servi ce

grant GM 35769 to J .G.B. f rom the Na t iona l Ins t i t u t es of Hea l th ,

a Nat ional Sci ence Foundat ion Graduate Fel lowship to S .A.E. ,

and funds f rom the Associa t ion pour l a Recherche sur l e Cancer

to P.B.

The publ icat ion costs of this ar t icle were defrayed in par t by

payment of page charges . Thi s ar t i c l e must t herefore be hereby

m a r k e d a d v e r t i s e m e n t i n a c c o r d a n c e w i t h 1 8 U S C se c t io n

1734 solely to indicate this fact .

R e f e r e n c e s

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Escherichia coh

that af fects the

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6 5 9 - 6 7 1 .

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stabi l it y) pro t e in and r ibonuclea se E are encoded by the sam e

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Escherichia coh. Proc. Natl. Acad. Sci.

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1-5.

Baga, M., M. Goransson, S. Normark, and B.E. Uhl in. 1988.

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of

E. coh

pi l i n gene express ion .

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52: 197-206.

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in

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[ompA]

for an in t egra l me m-

brane pro t e in of

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pufLMX

m R N A i n

Rhodobacter capsulatus

i s i n i t i a t ed by non- r an-

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An in t er c i s t ronic s t em- loop s t ruc ture funct ions as an

mRNA decay t erminator necessary but i nsuf f i c i ent for

puf

m R N A s t a b i l i t y .

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Escherichia coli ompA

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br idge , Massachuset t s .

Emory, S.A. and J.G. Belasco. 1990. The

ompA

5 ' u n t r a n s l a t e d

R N A se g m e n t f u n c ti o n s i n

Escherichia coli

as a growth- r a t e-

r egula t ed mRNA st abi l i zer whose ac t iv i ty i s unre l a t ed to

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