Bioscience Reports i, 571-574 (1981)
Printed in Great Britain
571
D i r e c t m e a s u r e m e n t of absolute suppressor e f f i c i e n c y
Vishvanath NENE and Robert E. GLASS
Department of Biochemistry, Queen's Medical Centre,
Clifton Boulevard, Nottingham NG7 2UH, U.K.
(Received 4 June 1981)
We have devised a system for measuring the degree of
t r a n s l a t i o n a l r e a d t h r o u g h past a n o n s e n s e m u t a t i o n
which is based upon t h e q u a n t i t a t i o n of t h e t w o
translation products, the suppressed polypeptide and the
nonsense fragment. The a b s o l u t e e f f i c i e n c y of f o u r
d i f f e r e n t a m b e r suppressors (Sul, Su2, Su3, and SuT)
has been determined at two unique amber sites in the
structural gene for the 8 subunit of Escherichia coli
R N A polymerase.
N o n s e n s e suppressors are mutant tRNA species capable of recognizing translational stop codons and, thus, of p r e v e n t i n g p r e m a t u r e
termination of polypeptide chain elongation (reviewed in reference 1).
For convenience, suppressor efficiency is often d e t e r m i n e d f r o m t h e
biological activity of the suppressed polypeptide alone. However, since
activity is dependent upon the suitability of the inserted amino acid,
t h e a b i l i t y of a suppressor tRNA to function at a particular site is
dictated by the nature of the a m i n o acid i n s e r t e d as well as t h e
e f f i c i e n c y o f its i n s e r t i o n .
To m e a s u r e t h e a b s o l u t e suppressor
e f f i c i e n c y - by which we m e a n t h e f r e q u e n c y of t r a n s l a t i o n a l
readthrough - it is necessary to quantitate both translation products of
the mutant gene, namely, the prematurely t e r m i n a t e d f r a g m e n t and
the suppressed polypeptide.
We have c h a r a c t e r i z e d two m u t a n t E s c h e r i c h i a
coli
strains
harbouring unique amber lesions in the C-terminal region of r/x~B, the
structural gene for the 8 subunit of RNA polymerase. They synthesize
N-terminal fragments of molecular weight 135 500 and 123 000 ( t h e
size of the w i l d - t y p e 8 subunit is calculated to be 150 61g daltons
from the DNA sequence ( 2 ) ) .
Since t h e m a j o r i t y of m o n o m e r i c
protein species in E. c o l i are smaller than 100 000 daltons (3), it is
p r a c t i c a l to q u a n t i t a t e both t h e 8 f r a g m e n t and t h e s u p p r e s s e d
p o i y p e p t i d e by electrophoretic separation of total cellular proteins in
SDS-polyacrylamide slab gels.
We thus have a system ideally suited
for m e a s u r i n g absolute suppressor efficiencies and the influence that
other factors such as a reading c o n t e x t m a y have on t r a n s l a t i o n a l
readthrough.
Methods
The genotypes of the rpoB amber strains are given in the legend to
Table 1. Lysates of Sul, Su2, Su3, and Su7 s u p p r e s s e d d e r i v a t i v e s
were prepared at 30~ as described previously except that a 3.0-min
9
The Biochemical Society
572
NENE & GLASS
Table i.
Synthesis of 8 subunits and 8 fragments in the presence
of four different amber suppressors
Sul~ Su2, Su39 and Su7 are the phenotypic symbols for suppressor alleles supD,
supE, supF, and supU, w h i c h insert serine~ glutamine~ tyrosine~ and gluta m i n e / t r y p t o p h a n (in the ratio 9:1; reference 9) at amber sites (i0,ii); sup
alleles are carried on F-prime plasmids.
The genogype of AJI is rpoB+ (Rif-s)
lacZ53(Am) rpsL (Str-r) argG metB fecal (12). AJS021-AJS027 and AJ6031-AJ6037
are the suppressed derivatives of AJS020 (AJlrpoBl2(Am)/FllOrpoB70 (Rif-r) and
A J 6 0 3 0 (AJlrpoB1603(Am)/F110rpoB70 (Rif-r), respectively (AJ5020 was formerly
called AJ52; reference 3); rpoB numbers are according to r e f e r e n c e
13.
Synthesis is expressed in terms of [3H]leucine incorporated as a percentage of
total labelled protein.
812 and 81603 refer to the 8 fragments synthesized by
rpoBl2 and rpoBl603 amber strains.
Strain (relevant genotype)
8 subunit
~ fragment
812
AJI (rpoB +)
0.30 • 0.02
-
AJ5021 (rpoBl2(Am) supD (Sul))
0.28 • 0.02
0.20 -+ 0.02
AJ5022 (rpoBl2(Am) supE (Su2))
0.17 -+ 0.01
0.49 • 0.02
81603
-
AJ5023 (rpoBl2(Am) supF (Su3))
0.40 -+ 0.02
0.18 • 0.01
AJ5027 (rpoBl2(Am) supU (SuT))
0.23 • 0.04
0.43 t 0.ii
AJ6031 (rpoBl603(Am) supD (Sul))
0.29 • 0.02
-
AJ6032 (rpoBl603(Am) supE (Su2))
0.14 z 0.02
-
1.05 -+ 0.20
AJ6033 (rpoB1603(Am) supF (Su3))
0.28 z 0.04
-
0.14 z 0.02
AJ6037 (rpoB1603(Am) supU (SuT))
0.32 + 0.02
-
0.30 • 0.04
0.49 • 0.04
p u l s e - a n d - c h a s e period was employed; such labelling conditions ensure
minimal degradation of labile proteins ( 3 ) .
Radiolabelled proteins
were fractionated on 2g-cm-long, 596 5DS-polyacrylamide slab gels (4)
at a constant current of 10 mA for 20 h.
The gel t r a c k s w e r e
e x c i s e d , a f t e r v i s u a l i z a t i o n of protein bands b y staining, and I-mm
slices counted for r a d i o a c t i v i t y as b e f o r e ( 4 ) .
The l a r g e r a m b e r
fragment has been previously characterized (3); further details on this
t r u n c a t e d p o l y p e p t i d e and t h e s m a l l e r s p e c i e s will be p r e s e n t e d
elsewhere.
R e s u l t s and D i s c u s s i o n
In s t r a i n s carrying amber lesions in rpoB, each productive translational initiation event on the B mRNA can give rise to e i t h e r a
c o m p l e t e 8 p o l y p e p t i d e or an N-terminal fragment, depending upon
whether there is translational readthrough past the nonsense site. The
e f f i c i e n c y of suppression of premature translational termination can,
therefore, be determined from the proportion of c o m p l e t e 8 c h a i n s
relative to the number of translational initiation events (i.e. the total
number of B and B-fragment molecules). The large amber fragments
synthesized by mutants carrying the rpoBl2 and rpoBl603 nonsense
lesions (Table i ) has allowed us to determine the absolute efficiency
of 5ul, Su2, Su3, and Su7 at two distinct sites (Table 2).
ABSOLUTE
SUPPRESSOR
Table 2.
EFFICIENCY
573
The absolute efficiency of suppresion
of rpoBl2(Am) and rpoBl603(Am)
Absolute suppressor efficiency (%)
Suppressor
rpoBl2(Am)
rpoBl603(Am)
58
26
69
34
37
12
66
52
Sul
Su2
Su3
Su7
Absolute
through)
suppressor
=
efficiency
(degree of translational read-
total number of suppression events
~otal number of translation initiations
Per cent total 31{ in
percent total 3H in 8 + percent total
3H in 8 fragment
x i00
(Note that initiation in this instance refers to events that occur
at the normal ~ ribosome binding site and not internal starts.)
The four amber suppressors vary in efficiency over about a six-fold
range (12 to 69%; Table 2). The fact t h a t Su3 is the most efficient
and Su2 t h e l e a s t e f f i c i e n t suppressor at the two mutant sites is
consistent with previous data (5). The ranking order of the suppresso r s , Su2 < Su7 < S u l < Su3 a n d Su2 < Sul < Su7 < Su3 for
rpoBl2(Am) and rpoB1603(Am), r e s p e c t i v e l y , d e m o n s t r a t e s t h e
i m p o r t a n c e of the r e a d i n g c o n t e x t for nonsense suppression. This
contention is f u r t h e r s u p p o r t e d by t h e d i f f e r e n c e in t r a n s l a t i o n a l
readthrough frequency for a single suppressor at the amber sites: only
the strongest suppressor, Su3, appears to function largely independently
of the reading context.
O u r s y s t e m is a m e n a b l e to a n a l y s i s of p a r a m e t e r s , such as
ribosomal discrimination (6), which can be imposed upon t h e t r a n s lational event.
For e x a m p l e , t h e s t r a i n s e m p l o y e d carry a zpsL
( S t r - r ) a l l e l e t h a t a p p e a r s to r e s t r i c t s u p p r e s s o r f u n c t i o n ( s e e
reference 7) at the readthrough level since addition of streptomycin to
the growth medium increases translational r e a d t h r o u g h (V. Nene &
R.E. Glass, m a n u s c r i p t in preparation).
Moreover, although it has
been possible to measure suppressor function in t h e p a s t by q u a n t i taring the relative activity of the suppressed polypeptide or its ability
to fulfil a structural role in a hetero-oligomer (see for instance 8),
such p r o c e d u r e s c a n n o t t a k e into c o n s i d e r a t i o n transcriptional or
t r a n s l a t i o n a l c o n t r o l s on gene e x p r e s s i o n .
M e a s u r e m e n t of both
translation products allows for regulatory effects (the implications of
the results on RNA polymerase control will be discussed elsewhere).
The s y s t e m d e s c r i b e d is a p p l i c a b l e only to the study of amber
suppressors.
We are at present in t h e p r o c e s s of o b t a i n i n g o c h r e
derivatives of these large-fragment-producing strains.
NENE & GLASS
574
Acknowledgements
We are most g r a t e f u l to all those who sent strains. We thank
Lepetit Pharmaceuticals Ltd., Maidenhead, for a g i f t of rifampJcin.
This work was supported by an MRC Project Grant.
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