- Email: [email protected]
The molecular weight of rabbit globin messenger RNA's
_v+
3
FEns Ce'rr
march 1973
THE MOLECULAR WEIGHT OF RABBIT GI.OBIN MESSENGER RNA'S HJ. GO~ and P.IL HAMLYN DeTm~ment o f BiOlahyslcf, King's Colle&e. 2 6 - 2 9 Drury Lane. London. WC2B 5RL. England Received 22 January 1973
I, Introduction
centrifuging this for I hr at 30,000 rpm (105,000 g) [5[. m R_NP (i.e. messenger-ribonucleic protein) was
Previous molecular weight measurement~ o f rabbit globln messenger RNA ( m R N A ) by polyacrylamide gel electrophoresis [ !, 21 have been performed in aqueous.systems in which the'secondary structure o f the molecules is conserved, in this study formamide was used-to dest~-oy all secondary structure [31 o f the
released from the polysomes by suspension in ! 0 mM Tris, 33 mM EDTA pH 7.4 and isolated by centrifuga. tion in a zonal rotor [61 (see fig. ! fo, details). T h e mRNP was precipitated from the gradient fractions by making them O. !% SDS, 0 ~ M NaCI then adding ~ vol ofethanoI. The precipitate was kept at - 2 0 ° for several hours and then the proteins removed by the
RNA (both mRNA and standards) so that conformati0nal changes do not affect the electrophozetic mobility and therefore the distance misrated in a gel by an RNA species is proportional to the log, molecular weight only.
phcnot/SDS method [71. After reprecipitating three times the final precipitate was washed twice with 7 0 ~ ethanol and dried in vacuo.
The RNA's used as standards had also been treated to ensure as complete re~noval o f proteins as possible 181. These were p2, 23 S, 16 S, 18 S, 5 S and 4 S.
2. Metlaoda 2.2. F o r m a m i d e gei electrophoresis The method used was based on that described by Stayno,I et al. [91. The following modifications were suggestc'd by J. Pinder (personal communication): the
2.t. Preparationo [ RNA A i),zate was made o f the red blood ceils from anaemic rabbits [4] and poly',,.omes obtained by -i
~
i
I
I
I
I
I
,----
0"6
0-4 0'2 I
!
I
I
I
30
:35
20
IS
I0
FRACTION
-..
I
5
~0
NUMBi~
Fig. I. p o l y l o m ~ t tmpended In 20 ml o f l 0 mM T d ~ - 3 3 mM E D T A . pH 7.4. were layered onto a suc~rose gradient, 1 0 ~ mtcrot, e l0 mM.TrI~'pH 7.4, to 40% ~ c r o ~ e - 1 0 m M T r i a , pH 7 A , then an overlay of~]0 nd !0 mM Tris was added. CentHfusation was for 16 In" at 48,.000 rpm and 4 ° in ~ Sptnco B X I V . Abbreviations: Hb = haemoglobin, mRNP = messenger ribonucleoproteln.
North.Hollamd P u b i ~ n g Company - Amsterdam
301
• ~/olume 30, a m b e r 3 :.~
,"
_
.
.-
-,-~
:taye . r ~ on.tOp, o f tier gel raider deionised buffered
fo . ~ ~ : ~ ¢ ~t~t~o~,~ r~o.o2 M N~,c~~,,~: ~ ¢~i[¢idated~betweentheconipor~mentt of the electro. I00 V,.10 mA- for 4.gels'(.7 ~ stained with
~ di~.eter). After eym
Y, o.5
acetic acid,- 1 mM citric, acid, amd de'stained with 0.5% .
i ~'
r
-
aceticacid (~. 2). 3. Resulls St ain.edgels were w a n n e d In a Gilford scanner so that the distance migrated b y each RNA from the orisln could be accurately measured. By plot tin 8 the distance migrated 2u~ninst log. molecular Jveight of the standard RNA'$ a stratsht line is~obtained From which the moleeular weight can be c a l c u l a t e d ( f i g . 3). The distance migrated by the same RNA species in different gels is n o t ahvays reproducible for r e a s o m not lofty u/~lerstood, For this reason the curves in'F~. 3 have different slopes, and internal markers must be used. As can be seen from fig. 2 the m R N A (which runs as one band in a q u e o u s gels [ 1 , 2 ] ) splits into two bands. These are labelled M 1 and M 2 (M I is the larger). M 1 has a molecular weight o f 2 2 7 , 0 0 0 (standard deviation 3. !%) and M 2 o f 2 0 2 , 0 0 0 (standard deviation 2.7%). See table 1 . I t is assumed that these bands rep[esent a~ and 1~ m~.NA xlnoe t l ~ RNA used was made From 14 S m R N P which has been s h o w n to be a purer source o f nul~,NA than 9 S RNA [16[. Kazazian
Te
F-~g. 2. Disc e l e c t r o p h o r e s ~ o f /~2 R N A , 24 S, ! 8 S, m R N A , 5 S a n d 4 S.
use o~"amberlite instead o[" Dowex to deionise the fom~amlde, buffering the f o r m a m i d e at pH 9 w i t h 0.02 M diethyl barbituric acid; the use Of disc gels rather than flat bed gels, 3.5% Polyacrylamide gels ( o f w h i c h i 5% was bisacrylamide) were used to separate a m i x t u r e o f mRNA and RNA's o f k n o w n molecular weisht. The
Table I mol~c~tf~rweight of zabbit haemoglobin messenger RHA's. E x o ~ i
Me~t~ured M W (8 determlnntions}
Expected MW*
Mt = p mRNA
227,000 SO = 3.1%
M2 ~ ¢~rnRNA
202,000 SD ~ 2.7%
.
_ E x McW e 's- s . t~-expected)
expaesu,:d In no. of nucleotides
140,160
86,840 SD '= 8.1%
271 SD = 8.1%
! 35,360
66,640 SD c 8.3%
280 SD ~ 8.3%
* Ex-;~cted MW calculsted using numbe~ of amino acida in clmln [ ISi, avmase nucleotlde MW of 320 and c.~mg ratio of 3 amino actd~/nucleotidc [ lgl. 302
!,Volu~ib~,30.pu'n..'bm" 3
FEeS LETTERS
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4 5 DISTANCE
1
I
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3
4
s
.
I
I.
i
I
i
i
2
3
4
s
F'-~. 3. Data used for the calculation o f the molecuLax weight of M, and M2. The standard~ used were (,n clegL'endin8 order) u2 I ~ (ea,sentialb/ the smote as RIT) [IO]. 23 S from E. c o l i I I I I. 18 S from zabblt reticuloLw'tes 112J. 16 S f r o m ~' co// lit 3J. 5 S It41 and 4 S [ 051 from rabbit retlculocytes. has shown t h a t m R N A f o r / 3 g l o b i n is larger than mRNA for at g l o b i n [17] a n d so M t is e q u a t e d w i t h
~ mP~A.
4. Discussion Other s t u d i e s o f r a b b i t h a e m o g l o b i n m R N A o n polyacrylamide gels h a v e c o n c e n t r a t e d e i t h e r o n measurin~g the m o l e c u l a r w e i g h t ( f o r w h i c h o n e value is obtabled), a t an a c r y l a m i d e c o n c e n t r a t i o n w h i c h allows internal s t a n d a r d s to b e i n c l u d e d [ I , 2[, o r d e m o n strati, g the h e t e r o g e n e i t y o f t h e R N A b y using a
higher gel co ncentrati0n [ 17]. The present study cornhines these t w o sinc~ in f o r m a m t d e t w o m R N A ' s
separate at a gel c o n c e n t r a t i o n suitable for use w i t h a wide range o f m a r k e r R N A ' s o f k n o w n m o l e c u l a r weight. In a d d i t i o n , this m e t h o d s h o u l d p r o v i d e increased a c c u r a c y in m o l e c u l a r w e i g h t d e t e r m i n a t i o n d u e to the removal o . f s e c o n d a r y s t r u c t u r e in t h e R N A . T h e results o b t a i n e d in these e x p e r i m e n t s are at the u p p e r e n d o f the range o f m o l e c u l a r w e i g h t s a l r e a d y p u b l i s h e d . C h a n t r e n n e et al. [201 c a l c u l a t e d a value o f 150,000 f r o m m e a s u r e m e n t s o f the s e d i m e r i t a t i o n coefficient. Using p o l y a c r y l a m i d e gels - a m o r e sensitive
method, Gaskill and Kabat [ I ! obtained a molecular weight o f 2 2 0 , 0 0 0 , L~brie [2] 1 9 0 , 0 0 0 and KaTazian 180,000 ( p e r s o n a l c o m m u n i c a t i o n ) .
There is a difference of about 3% in the number of amino acids in the two globin chains (~ has 146 and 303
Volume 30; nu~itber 3
a 141 [18l). ut m ~ h t be suppos~,thaLZ the mRN_A's would reflect this difference. However, Ka~zJ~m . reports a 5% difference and we find it is about 10%. This corresponds to an extra 6 0 n u c l e o t i d e s whet:e~s the expected difference is 15. Lira and CaneHakts [21] have reported that a region o f rabbit globin mRNA between 50 and 70 nucleoUdes long is oitef 70% adenine..This 'poly-A" rich region i s ' t h o t ~ t unllkely to be informational [22]. Their experiments do not distinguish between a and fl mRNA although they observe that there is only about half as much poly-A in mRNA obtained from lishter p o l y ~ m e s . It is known that in lighter polysomes there i~ more or m R N A than /] m R N A [23']. Therefore it is possible that or mRNA contains appre,_~iably less poly-A and that fl mRNA is Larger b e c a u s e it c o n ' t a i n s this a d e n i n e rich s e q u e n c e .
Acknowledgements We s h o u l d like t o t h a n k Dr. H. l s e n b e r g f o r a gift o f
/J2 RNA and J. Pinder for gifts o f 23 S and 16.S RNA and for valuable advice in the method o f polyacrylamide gel e l e c t r o p h o r e s i s o f R N A in f o r m a m i d e . H . J . G . is a
recipient o f M.R.C. research grant G 969/509/B. P.H.tL holds an M.R.C. research studentship.
Re(erences [ 11 P. Gask3,11and D. Kabat, Proc. NaIL Acad. Sct. U.S. 68 (1971) 72. [2[ F. Labde. Nature 221 (1969) 1217. [3] P.O.P. Ts'o, G.H. HeEmkamp and C. Sander. Biochen~ Biophys. Acta 55 (1962) 584.
304
14| H.R.V.~~ "'
R.A. Cox iu~l7.A".H~uat, B i m : ~ l ~ J.
"" 9 2 ( I ~ 4 ) ' 6 4 8 . " "
/
iSl M.L -.42p.
:
'.~
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-,
20 O971)
- ".
161 R- wm~,~. ,m~ M. Mort'hub'n, G, _l~ny~n and J. Paul, BiochemiJtry IO (1971)"3014,
I7( t~.w. Nkimbem and J.H. Mailme~ ~ •
[81 [9]
[10] [ 1i ] [12] [131 •
Natt. Acad. S,Y,.
U.S. 47 (1961 | 1588. .. K.S. KWoy;B!0chem. J. 96 (1965) 7~26. DJ.'Staynov, J.C. Pinder, W.B. GratzeG Nature New nio]~u~ 235 ( 1 9 7 2 ) ms. M.D. Enser, E.A. Stubb~ S. MIU'a and P, K ~ Prec. Natl. A c a d . S c L U-S. 49 (1963) 857. G.C. KurlLandL,J. MoL Biol. 2 (1966) 83. W.M. Stanley and ILM. Bock, Biochemistry 4 (1965) ! 302. E.H. Mc~C?onlk©ya ~ l J.W. Hopkin~ J. Mok BioL 39 (1968) 5 4 5 . B. ForKn:t and ~. Weismnan, Science 158 (1967) i695.
['14] [15] Molecular weight taken ~ 28,000, E. RI 4mrds, pcrsonaJ
communication. [16] W.G. Lanyon, J. Pauland R. Wtllimmmn. European J. Biochem. 31 (1972) 38. [ ! 71 H.H. KaTp~!an~ Nature 238 ( 197 !) 166. 1! 81 From the "Atlas o f Protein Sequemce and Structure" 1967--68, R-V. Eck and M.O. Dayhoff, published by The NaUonal Biomedical Research F o u n d a t i o n [19[ C.R. W ~ !~. Nuc.. Acid Res. Mol. BioL 7 (196/) 107. [20[ H. Chanlrenne, A, Burny and G. blarbaix, Pmgr. Nuc. Acid Res. ModL~BiolL 7 (1967) 173. 1211 L. tam and E.S. Canel/ak~ Nature 227 (1970) 710, [22] J.B.I.tn~-el, R.E. Lockard, R.F. Jonet, H.E_ Burr and l.W. HoMer, ~ Haematologica 4 (1971) 37. 1231 R.T. Hunt, A.R. Hunter. A.J. Munro, Nature 220 (196.8) 481.
3
FEns Ce'rr
march 1973
THE MOLECULAR WEIGHT OF RABBIT GI.OBIN MESSENGER RNA'S HJ. GO~ and P.IL HAMLYN DeTm~ment o f BiOlahyslcf, King's Colle&e. 2 6 - 2 9 Drury Lane. London. WC2B 5RL. England Received 22 January 1973
I, Introduction
centrifuging this for I hr at 30,000 rpm (105,000 g) [5[. m R_NP (i.e. messenger-ribonucleic protein) was
Previous molecular weight measurement~ o f rabbit globln messenger RNA ( m R N A ) by polyacrylamide gel electrophoresis [ !, 21 have been performed in aqueous.systems in which the'secondary structure o f the molecules is conserved, in this study formamide was used-to dest~-oy all secondary structure [31 o f the
released from the polysomes by suspension in ! 0 mM Tris, 33 mM EDTA pH 7.4 and isolated by centrifuga. tion in a zonal rotor [61 (see fig. ! fo, details). T h e mRNP was precipitated from the gradient fractions by making them O. !% SDS, 0 ~ M NaCI then adding ~ vol ofethanoI. The precipitate was kept at - 2 0 ° for several hours and then the proteins removed by the
RNA (both mRNA and standards) so that conformati0nal changes do not affect the electrophozetic mobility and therefore the distance misrated in a gel by an RNA species is proportional to the log, molecular weight only.
phcnot/SDS method [71. After reprecipitating three times the final precipitate was washed twice with 7 0 ~ ethanol and dried in vacuo.
The RNA's used as standards had also been treated to ensure as complete re~noval o f proteins as possible 181. These were p2, 23 S, 16 S, 18 S, 5 S and 4 S.
2. Metlaoda 2.2. F o r m a m i d e gei electrophoresis The method used was based on that described by Stayno,I et al. [91. The following modifications were suggestc'd by J. Pinder (personal communication): the
2.t. Preparationo [ RNA A i),zate was made o f the red blood ceils from anaemic rabbits [4] and poly',,.omes obtained by -i
~
i
I
I
I
I
I
,----
0"6
0-4 0'2 I
!
I
I
I
30
:35
20
IS
I0
FRACTION
-..
I
5
~0
NUMBi~
Fig. I. p o l y l o m ~ t tmpended In 20 ml o f l 0 mM T d ~ - 3 3 mM E D T A . pH 7.4. were layered onto a suc~rose gradient, 1 0 ~ mtcrot, e l0 mM.TrI~'pH 7.4, to 40% ~ c r o ~ e - 1 0 m M T r i a , pH 7 A , then an overlay of~]0 nd !0 mM Tris was added. CentHfusation was for 16 In" at 48,.000 rpm and 4 ° in ~ Sptnco B X I V . Abbreviations: Hb = haemoglobin, mRNP = messenger ribonucleoproteln.
North.Hollamd P u b i ~ n g Company - Amsterdam
301
• ~/olume 30, a m b e r 3 :.~
,"
_
.
.-
-,-~
:taye . r ~ on.tOp, o f tier gel raider deionised buffered
fo . ~ ~ : ~ ¢ ~t~t~o~,~ r~o.o2 M N~,c~~,,~: ~ ¢~i[¢idated~betweentheconipor~mentt of the electro. I00 V,.10 mA- for 4.gels'(.7 ~ stained with
~ di~.eter). After eym
Y, o.5
acetic acid,- 1 mM citric, acid, amd de'stained with 0.5% .
i ~'
r
-
aceticacid (~. 2). 3. Resulls St ain.edgels were w a n n e d In a Gilford scanner so that the distance migrated b y each RNA from the orisln could be accurately measured. By plot tin 8 the distance migrated 2u~ninst log. molecular Jveight of the standard RNA'$ a stratsht line is~obtained From which the moleeular weight can be c a l c u l a t e d ( f i g . 3). The distance migrated by the same RNA species in different gels is n o t ahvays reproducible for r e a s o m not lofty u/~lerstood, For this reason the curves in'F~. 3 have different slopes, and internal markers must be used. As can be seen from fig. 2 the m R N A (which runs as one band in a q u e o u s gels [ 1 , 2 ] ) splits into two bands. These are labelled M 1 and M 2 (M I is the larger). M 1 has a molecular weight o f 2 2 7 , 0 0 0 (standard deviation 3. !%) and M 2 o f 2 0 2 , 0 0 0 (standard deviation 2.7%). See table 1 . I t is assumed that these bands rep[esent a~ and 1~ m~.NA xlnoe t l ~ RNA used was made From 14 S m R N P which has been s h o w n to be a purer source o f nul~,NA than 9 S RNA [16[. Kazazian
Te
F-~g. 2. Disc e l e c t r o p h o r e s ~ o f /~2 R N A , 24 S, ! 8 S, m R N A , 5 S a n d 4 S.
use o~"amberlite instead o[" Dowex to deionise the fom~amlde, buffering the f o r m a m i d e at pH 9 w i t h 0.02 M diethyl barbituric acid; the use Of disc gels rather than flat bed gels, 3.5% Polyacrylamide gels ( o f w h i c h i 5% was bisacrylamide) were used to separate a m i x t u r e o f mRNA and RNA's o f k n o w n molecular weisht. The
Table I mol~c~tf~rweight of zabbit haemoglobin messenger RHA's. E x o ~ i
Me~t~ured M W (8 determlnntions}
Expected MW*
Mt = p mRNA
227,000 SO = 3.1%
M2 ~ ¢~rnRNA
202,000 SD ~ 2.7%
.
_ E x McW e 's- s . t~-expected)
expaesu,:d In no. of nucleotides
140,160
86,840 SD '= 8.1%
271 SD = 8.1%
! 35,360
66,640 SD c 8.3%
280 SD ~ 8.3%
* Ex-;~cted MW calculsted using numbe~ of amino acida in clmln [ ISi, avmase nucleotlde MW of 320 and c.~mg ratio of 3 amino actd~/nucleotidc [ lgl. 302
!,Volu~ib~,30.pu'n..'bm" 3
FEeS LETTERS
Ma~ch 19"/3
r
i0 7
i, , .
~i ~ '
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- ~ ~ . "
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I
F
I
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1041
I
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5
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.|
4 5 DISTANCE
1
I
I
I
i 2 (CM)
3
4
s
.
I
I.
i
I
i
i
2
3
4
s
F'-~. 3. Data used for the calculation o f the molecuLax weight of M, and M2. The standard~ used were (,n clegL'endin8 order) u2 I ~ (ea,sentialb/ the smote as RIT) [IO]. 23 S from E. c o l i I I I I. 18 S from zabblt reticuloLw'tes 112J. 16 S f r o m ~' co// lit 3J. 5 S It41 and 4 S [ 051 from rabbit retlculocytes. has shown t h a t m R N A f o r / 3 g l o b i n is larger than mRNA for at g l o b i n [17] a n d so M t is e q u a t e d w i t h
~ mP~A.
4. Discussion Other s t u d i e s o f r a b b i t h a e m o g l o b i n m R N A o n polyacrylamide gels h a v e c o n c e n t r a t e d e i t h e r o n measurin~g the m o l e c u l a r w e i g h t ( f o r w h i c h o n e value is obtabled), a t an a c r y l a m i d e c o n c e n t r a t i o n w h i c h allows internal s t a n d a r d s to b e i n c l u d e d [ I , 2[, o r d e m o n strati, g the h e t e r o g e n e i t y o f t h e R N A b y using a
higher gel co ncentrati0n [ 17]. The present study cornhines these t w o sinc~ in f o r m a m t d e t w o m R N A ' s
separate at a gel c o n c e n t r a t i o n suitable for use w i t h a wide range o f m a r k e r R N A ' s o f k n o w n m o l e c u l a r weight. In a d d i t i o n , this m e t h o d s h o u l d p r o v i d e increased a c c u r a c y in m o l e c u l a r w e i g h t d e t e r m i n a t i o n d u e to the removal o . f s e c o n d a r y s t r u c t u r e in t h e R N A . T h e results o b t a i n e d in these e x p e r i m e n t s are at the u p p e r e n d o f the range o f m o l e c u l a r w e i g h t s a l r e a d y p u b l i s h e d . C h a n t r e n n e et al. [201 c a l c u l a t e d a value o f 150,000 f r o m m e a s u r e m e n t s o f the s e d i m e r i t a t i o n coefficient. Using p o l y a c r y l a m i d e gels - a m o r e sensitive
method, Gaskill and Kabat [ I ! obtained a molecular weight o f 2 2 0 , 0 0 0 , L~brie [2] 1 9 0 , 0 0 0 and KaTazian 180,000 ( p e r s o n a l c o m m u n i c a t i o n ) .
There is a difference of about 3% in the number of amino acids in the two globin chains (~ has 146 and 303
Volume 30; nu~itber 3
a 141 [18l). ut m ~ h t be suppos~,thaLZ the mRN_A's would reflect this difference. However, Ka~zJ~m . reports a 5% difference and we find it is about 10%. This corresponds to an extra 6 0 n u c l e o t i d e s whet:e~s the expected difference is 15. Lira and CaneHakts [21] have reported that a region o f rabbit globin mRNA between 50 and 70 nucleoUdes long is oitef 70% adenine..This 'poly-A" rich region i s ' t h o t ~ t unllkely to be informational [22]. Their experiments do not distinguish between a and fl mRNA although they observe that there is only about half as much poly-A in mRNA obtained from lishter p o l y ~ m e s . It is known that in lighter polysomes there i~ more or m R N A than /] m R N A [23']. Therefore it is possible that or mRNA contains appre,_~iably less poly-A and that fl mRNA is Larger b e c a u s e it c o n ' t a i n s this a d e n i n e rich s e q u e n c e .
Acknowledgements We s h o u l d like t o t h a n k Dr. H. l s e n b e r g f o r a gift o f
/J2 RNA and J. Pinder for gifts o f 23 S and 16.S RNA and for valuable advice in the method o f polyacrylamide gel e l e c t r o p h o r e s i s o f R N A in f o r m a m i d e . H . J . G . is a
recipient o f M.R.C. research grant G 969/509/B. P.H.tL holds an M.R.C. research studentship.
Re(erences [ 11 P. Gask3,11and D. Kabat, Proc. NaIL Acad. Sct. U.S. 68 (1971) 72. [2[ F. Labde. Nature 221 (1969) 1217. [3] P.O.P. Ts'o, G.H. HeEmkamp and C. Sander. Biochen~ Biophys. Acta 55 (1962) 584.
304
14| H.R.V.~~ "'
R.A. Cox iu~l7.A".H~uat, B i m : ~ l ~ J.
"" 9 2 ( I ~ 4 ) ' 6 4 8 . " "
/
iSl M.L -.42p.
:
'.~
E zymot " "
-,
20 O971)
- ".
161 R- wm~,~. ,m~ M. Mort'hub'n, G, _l~ny~n and J. Paul, BiochemiJtry IO (1971)"3014,
I7( t~.w. Nkimbem and J.H. Mailme~ ~ •
[81 [9]
[10] [ 1i ] [12] [131 •
Natt. Acad. S,Y,.
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['14] [15] Molecular weight taken ~ 28,000, E. RI 4mrds, pcrsonaJ
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