DIE NATURWISSENSCHAFTEN 63. Jahrgang
Heft8
August 1976
Initiation of Protein Synthesis Severo Ochoa '~ Roche Institute of Molecular Biology, Nutley, New Jersey 07110, U.S.A.
This lecture deals with the mechanism of polypeptide chain initiation in bacteria and other prokaryotic cells, specially with chain initiation and with the nature and function of the initiation factors. Our knowledge of how ribosomes recognize messenger initiation sites is also reviewed.
Protein synthesis is the last step in expression of genetic information. In this step, messenger R N A is translated to yield a polypeptide chain whose amino acid sequence is determined by the nucleotide sequence of the messenger. There are three steps in translation: initiation, elongation, and termination. Chain initiation and the beginning of chain elongation are schematically represented in Figure 1. In prokaryotes the mRNA is first bound to the small ribosomal subunit. This is followed (step 1) by binding of the initiator aminoacyl tRNA to the small subunit-mRNA complex. This step requires Mg 2+, initiation factors (IF), and GTP. Next, the larger ribosomal subunit joins this complex to form the initiation complex proper (step 2) and the stage is set for chain elongation. The second aminoacyl-tRNA is bound in a reaction (step 3) that requires GTP and an elongation factor (EF-1) whereby GTP is hydrolyzed to GDP and Pi. The first (initiator) and second aminoacyl-tRNA occupy sites on the ribosome designated as the peptidyl (P) and aminoacyl (A) site, respectively. Peptide bond formation takes place (reaction 4); the A site now bears dipeptidyl-tRNA while the P site bears the deacylated initiator tRNA. In the next (translocation) step (step 5)the ribosome is displaced relative to t h e messenger-peptidyl tRNA complex; the initiator tRNA is ejected and the peptidyl tRNA is now on the P site. This sets the stage for binding of the third aminoacyl tRNA and these processes are repeated until the polypeptide chain is completed. Translocation requires a second elongation factor (EF-2) and one more GTP which is hydrolyzed to GDP and Pi. Whereas the steps and mechanisms of protein syn* Lecture held on June 20, 1975 as the 15th Karl August Forster Lecture on the Akademie der Wissenschafteu und der Literatur Mainz and the Arbeitsgruppe Programmierte Synthese der J o h a n nes Gutenberg-Universit~t Naturwissenschaften 63, 347-355 (1976)
thesis are basically identical in prokaryotes and eukaryotes, there has occurred considerable evolutionary change of the proteins involved, both ribosomal and soluble, probably related to the increasing need for translational control in nucleated cells. Thus, whereas ribosomal subunits or factors from different species can readily be exchanged within each domain, there is little or no hybridization across the prokaryoticeukaryotic cytoplasmic line. From Figure 1, one may get the impression that protein is synthesized on the ribosomal surface, but this is not so. Synthesis occurs aa-tR N~ (Mg2+,TF, GTP)
,'nRNA
oo- tRNA2+ G T P ~ , (EF-1) 3
GOP + P~,~/
5
4
GDP + Pi -EIRNA~ GYP (EF-2)
Fig. I. Reactions involved in polypeptide chain initiation and elongation [1]
30 S
50 S
r Fig. 2. Three-dimensional representation of the translating E. coli ribosome. Initiator (fMet-tRNAf) and mRNA are shown bound to the contacting (inner) surface of the 30S and the second aminoacyl-tRNA (lys-tRNA) to the contacting surface of the 50S subunit. L P, and A represent the initiation, peptidyl, and aminoacyl binding sites. When the ribosome locks, the A and P sites overlap [2]
9 by Springer-Verlag 1976
347
at the interface between the two ribosomal subunits as depicted in Figure 2 for prokaryotic ribosomes.
cleotides such as A U G A A A A A A . . . A A A ( A U G A , ) , beginning with an initiatior codon A U G at the 5' end, yielded Met-Lys-Lys...Lys oligopeptides with methionine (Met) at the amino-terminal end, the remaining amino acid residues being all lysine (Lys) residues. Thus, a small a m o u n t of [t4C]methionine and a larger a m o u n t of [~4C]lysine was incorporated into acid-insoluble products. When similar oligonucleotides but beginning with a non-initiator codon (e.g., the glycine codon, G G U ) were used ( G G U A , ) their translation required a higher Mg 2+ concentration and, in any case, the non-initiating codon was not translated; the resulting peptides contained only [14C]lysine [4]. As seen in Figure 3, the addition of initiation factors IF-1 and IF-2, partially purified f r o m the ribosomal wash, was essential for translation of oligonucleotides beginning with an A U G codon at the 5' end, whether at 14 m M or 18 m M Mg 2+. On the other hand, oligonucleotides beginning with G G U which were not translated at 1 4 r a M Mg 2§ with or without factors, were translated at 18 m M Mg 2 + in the absence of added factors. Table 1 summarizes some of the early results of our laboratory [3, 5]. At first, we washed the ribosomes with 0.5 M NH4C1 and, utilizing the f M e t - t R N A r ribosomal binding assay referred to below, isolated and partially purified two protein factors, IF-1 and IF-2. These factors (Table 1, experiment 1) were not required for translation of poly(A) and their addition produced n.o stimulation. However, translation of natural messengers, such as the R N A s of the coilphages MS2 and Q/~, required addition of the factors. It was later found that, when the ribosomes were washed with 1.0 M NH4C1, one other factor, IF-3, was required for natural messenger translation (Table 1, experiment 2). Ribosomes can bind fMet-tRNAf, the initiator aminoacyl-tRNA, to form an initiation complex upon incubation in the presence of the trinucleoside-diphosphate A p U p G ( A U G ) as messenger. Ribosomal binding of f[14C]Met-tRNA is readily measured because the ribosome-bound, but not the free c o m p o u n d is retained by nitrocellulose filters following incubation
Initiation Factors: Nature and Function
The polypeptide chain initiation factors were discovered in 1965 through the use of E s c h e r i c h i a coil ribosomes that had been washed with concentrated salt solutions. These ribosomes, while translating synthetic messengers, such as poly(U) or poly(A), were unable to translate natural messengers unless the ribosomal wash was added back. Washed ribosomes were also able to translate oligonucleotide messengers that had non-initiating codons at the 5' terminus, but those having an initiator codon A U G in this position, required addition of the ribosomal wash for translation [3]. This finding was essential for establishing that the factors present in the ribosomal wash were in fact concerned with initation. Translation of oligonu-
1400 _~1200
-
tO
+Z -F
[']kysine
I
E
=
F=initiotion foctors Methionine
800-
O n
8
I 600
I
-o
'~ 400
E
o
200
0
-F l AUGA18GGUA2~AUGAIBGGUA2~ 14ml Mg2+ 18mM Mg2+
Fig. 3. Effect of initiation factors and Mg2+ on the translation of the oligoribonucle0tides AUGAn and GGUAn. A nuclease-low cell-free system,consisting of NH4Cl-washed E. coli Q13 ribosomes and LactobacilIus arabinosus high-speed supernatant was used. Incubation 40 min at 37 ~ +F, addition of initiation factors (IF-1 and IF-2); - F , no factor additions (prepared from Table 1 of [4])
Table 1. Effect of initiation on translation of natural messengers by NH4Cl-washed E. coli ribosomes (from [3, 5]). The translation system consisted of washed ribosomes and high-speed supernatant from E. coli Q13. The Mg2+ concentration was 18 mM for poly (A) translation and 14 mM in all other cases. Incubation was at 37 ~ for 40 min in experiment 1 and 20 min in experiment2 Exp. No.
Washingof ribosomes
mRNA
0,5 M NH4CI
Poly (A)
Q~ RNA
348
Amino acid incorporation [pmotes/sample] Lysine
MS2 RNA
1.0 M NH4C1
Factor additions
MS2 RNA
None IF-1 +IF-2 None IF- i + IF-2 None IF-I +IF-2 None Ribosomal wash IF-1 +IF-2 IF-3 IF-1 +IF-2+IF-3
4,898 4,518 64 606 332 1,200 20 980 45 355 1,082
Leucine
Methionine Histidine
68 767
15 155
Naturwissenschaften 63, 347-355 (1976)
10 92
9 by Springer-Verlag 1976
r-~
A.
u m
o 12 E
,=,
B.
fMet - tRNAf + I F - I + TF-2
"o
Q 1 0
= I0 :=
E
IF-I +IF-2
o "o
,Z
c
z
8
o .o
r,-
L :S
6
z er
,q, im
I
I
4
IF-2
7
only
2 I~"
Met-tRNAf
I
with I F - I
+TF-2
0 2
0
4 MINUTES
6
8
O 0
I0
2
4 6 MINUTES
8
Fig. 4. (A) Effect of IF-1 +IF-2 on the AUG-dependent binding of formylated and non-formylated fMet-tRNAf to E. coli ribosomes (from [4]). (B) Effect of IF-1 and IF-2, singly and in combination, on the AUG-dependent ribosomal binding of fMet-tRNAf (from [6]). Reactions were conducted at 5 mM Mg z+ and 25 ~
Table 2. AUG-dependent binding of fMet-tRNA r with ribosomal subunits at 0 ~ and 25 ~ (from [71). The ribosomal binding of t~C]Met-tRNAf was determined by the nitrocellulose filter procedure after incubation for 15 min in the presence of 3.5 mM Mg z§ 0.3 m M GTP, and AUG as messenger, at the indicated temperature Temp. [~
0
Subunits
30S 30S + 50S
25
30S 30S + 50S
Factor additions [ g g ] IF-2
IF- 1
f[14C]MettRNAf bound [moles]
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
None 1.0 None 1.0 None 1.0 None 1.0
1.4 1.7 4.0 8.0 0.4 1.9 0.7 4.8
Stimulation by IF-1
x 1.2 x2.0 x4.7 x6.8
in the presence of Mg 2+. Figure 4A shows the requirement of IF-1 and IF-2 for binding at 3 m M Mg 2 +. Under these conditions, non-formylated MettRNAf is not bound whether in the presence or absence of factors. In Figure 4B it may be seen that both IF-1 and IF-2 were needed for maximal ribosomal binding of f[~4C]Met-tRNAf and that dependence on IF-2 was complete. However, although there was a marked decrease in binding in the absence of IF-l, the requirement for this factor was not absolute. Is IF-1 an Initiation Factor?
When the binding is carried out a low temperature the dependence on IF-1 is markedly decreased. Table 2 shows that, when the temperature of incubation was dropped to 0 ~ there was good binding in the presence of IF-2 alone, whether with 30S or with 30S+50S subunits. At this temperature IF-1 Naturwissenschaften 63, 347-355 (1976)
produced only a moderate increase (up to two-fold) in binding. However, at 25 ~ the stimulation by IF-1 was five to seven-fold. These results suggest that IF-1 merely increased the stability of the initiation complex which is lower at the higher temperature [7]. Thus, IF-1 might be a component protein of the 30S initiation site which, unlike other ribosomal proteins, is readily extracted from E. coli ribosomes by salt washing. The view that IF-1 may be a ribosomal protein rather than an initiation factor is strengthened by the finding of Leffler and Szer in our laboratory [8] that no IF-Mike proteins were obtained by salt-washing of Caulobacter crescentus ribosomes. Moreover, in marked contrast to E. coli ribosomes, the AUG-directed, IF-2-dependent binding of fMet-tRNAe to C. crescentus ribosomes was as good at 24 ~ as at 0 ~ (Table 3, experiment A). It may also be seen (Table 3, experiment B) that addition of E. coli IF-1 caused no stimulation of initiation complex formation with C. crescentus ribosomes and C. crescentus phage Cb5 RNA at 37 ~ whereas the factor markedly stimulated complex formation by E. coli ribosomes with the E. coli-specific MS2 phage RNA. Recycling of IF-2, i.e., the alternating association and dissociation of the factor to and from ribosomes during protein synthesis (see below) may depend on the integrity of the 30S initiation site. This might explain why IF-1 is required for IF-2 recycling [9]. IF-1 has been isolated in crystalline form [10]; it is a small basic protein (molecular weight 9,400). It contains all of the common 20 amino acids with only one residue each of histidine, proline, cysteine, methionine, and tryptophan, and has alanine and lysine, respectively, at the amino and carboxy terminal ends. The amino acid composition of IF-1 is similar to that of the majority of ribosomal proteins which, although differing widely in molecular weight, are remarkably similar in amino acid composition.
9 by Springer-Verlag 1976
349
IF-2 is indispensable for formation of the 30S initiation complex and appears to recognize and promote the ribosomal binding of only one species of naturally occurring aminoacyl-tRNA, namely, the initiator fMet-tRNAf. In this regard, its function is similar to that of the chain elongation factor Tu which does not recognize the initiator but does recognize and promote the binding of all of the other aminoacyl tRNAs to the 70S ribosomal complex. IF-2 functions catalytically [12]. One molecule of IF-2 can promote the binding of several molecules of fMet-tRNA to ribosomes. Since IF-2 itself is bound to the 30S ribosomal subunit when the 30S complex is formed, it must be eventually released to become available for new rounds of initiation. This happens when the 50S subunit joins the 30S complex to form the 70S initiation complex (Fig. 1, step 2). This recycling of IF-2 requires GTP. No recycling is possible when the analog 5'-guanylylmethylene diphosphonate (GMPPCP), that cannot be hydrolyzed, is substituted for GTP, or when the 70S initiation complex is formed with
Table 3. Initiation factor requirements for formation on initiation complex with Caulobacter crescentus and Escherichia coli ribosomes (from [8]). Binding measured as in Table 2. A. Incubation was for 15 min at 24 ~ and 45 min at 0 ~ B. Incubation was for 10 min at 37 ~ A (Template, AUG) Source of washed ribosomes
pmoles of [l'~C]Met-tRNAfbound in the presence of C. crescentus IF-2
C. crescentus
ribosomal wash
C. crescentus E. coli E. coli+ IF-1
at0~
at 24~
at0~
at 24~
3.9 3.0
3.8 0.7 4.0
3.8 3.5
3.6 1.0 4.5
B (Template, natural mRNA) Source of washed ribosomes
mRNA
C. crescentus
Phage Cb5
IF-1
(C.crescentus)
(E. coli) tRNAf
f[a*c]Metbound [pmoles]
Phage MS2
E. coli
IF-2
"70 S 50 S
7_
-
0.3
/
-
+ ~-
+
/
-
+
+
2.4 2.3 0.1 0.3 2.9
I 70-/-g0X--1
-
:50 S
0"8
0"6
0.4
I(B) 7os
sos
|to 0.2
8 0-8
6
0-6
4 dr"
0-4
2
I
0
I0
20
30 0
I0
20
0.2
0
I
50
Fraction number Fig. 5. Interaction of 3aP-labelIed tF-2 with ribosomes. A 30S initiation complex was first formed with f[3H]Met-tRNAf ( e - - e ) and [32p]IF-2 ( o - - o), in the presence of either GMPPCP or GTP, by incubation for a further 5 min the samples were analyzed by sucrose density gradient centrifugation. (A) with GMPPCP and (B) with GTP (from [11])
0.8 --
06
-
,~
g)
0"4
IF-2 0-2
--
E. coli IF-2 has been purified to near homogeneity
in several laboratories [11]. The factor appears to exist in a t least two forms, IF-2a and IF-2b of molecular weights around 80,000 and 90,000, respectively. IF;2 is sensitive to sulfhydryl binding reagents such as p-mercuribenzoate or N-ethylmaleimide. 350
6. DF activity of IF-3. E. coli ribosomes with a high proportion of 70S couples were incubated for 20 min at 37 ~ (6.5 mM Mg 2 +) with n o factors (A), IF:I (B), or IF-3 (C), and the distribution of 30S, 50S, and 70S ribosomes was analyzed by sucrose gradient centrifngation (from [16]) Fig.
Naturwissenschaften 63, 347--355 (1976)
9 by Springer-Verlag 1976
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Heft8
August 1976
Initiation of Protein Synthesis Severo Ochoa '~ Roche Institute of Molecular Biology, Nutley, New Jersey 07110, U.S.A.
This lecture deals with the mechanism of polypeptide chain initiation in bacteria and other prokaryotic cells, specially with chain initiation and with the nature and function of the initiation factors. Our knowledge of how ribosomes recognize messenger initiation sites is also reviewed.
Protein synthesis is the last step in expression of genetic information. In this step, messenger R N A is translated to yield a polypeptide chain whose amino acid sequence is determined by the nucleotide sequence of the messenger. There are three steps in translation: initiation, elongation, and termination. Chain initiation and the beginning of chain elongation are schematically represented in Figure 1. In prokaryotes the mRNA is first bound to the small ribosomal subunit. This is followed (step 1) by binding of the initiator aminoacyl tRNA to the small subunit-mRNA complex. This step requires Mg 2+, initiation factors (IF), and GTP. Next, the larger ribosomal subunit joins this complex to form the initiation complex proper (step 2) and the stage is set for chain elongation. The second aminoacyl-tRNA is bound in a reaction (step 3) that requires GTP and an elongation factor (EF-1) whereby GTP is hydrolyzed to GDP and Pi. The first (initiator) and second aminoacyl-tRNA occupy sites on the ribosome designated as the peptidyl (P) and aminoacyl (A) site, respectively. Peptide bond formation takes place (reaction 4); the A site now bears dipeptidyl-tRNA while the P site bears the deacylated initiator tRNA. In the next (translocation) step (step 5)the ribosome is displaced relative to t h e messenger-peptidyl tRNA complex; the initiator tRNA is ejected and the peptidyl tRNA is now on the P site. This sets the stage for binding of the third aminoacyl tRNA and these processes are repeated until the polypeptide chain is completed. Translocation requires a second elongation factor (EF-2) and one more GTP which is hydrolyzed to GDP and Pi. Whereas the steps and mechanisms of protein syn* Lecture held on June 20, 1975 as the 15th Karl August Forster Lecture on the Akademie der Wissenschafteu und der Literatur Mainz and the Arbeitsgruppe Programmierte Synthese der J o h a n nes Gutenberg-Universit~t Naturwissenschaften 63, 347-355 (1976)
thesis are basically identical in prokaryotes and eukaryotes, there has occurred considerable evolutionary change of the proteins involved, both ribosomal and soluble, probably related to the increasing need for translational control in nucleated cells. Thus, whereas ribosomal subunits or factors from different species can readily be exchanged within each domain, there is little or no hybridization across the prokaryoticeukaryotic cytoplasmic line. From Figure 1, one may get the impression that protein is synthesized on the ribosomal surface, but this is not so. Synthesis occurs aa-tR N~ (Mg2+,TF, GTP)
,'nRNA
oo- tRNA2+ G T P ~ , (EF-1) 3
GOP + P~,~/
5
4
GDP + Pi -EIRNA~ GYP (EF-2)
Fig. I. Reactions involved in polypeptide chain initiation and elongation [1]
30 S
50 S
r Fig. 2. Three-dimensional representation of the translating E. coli ribosome. Initiator (fMet-tRNAf) and mRNA are shown bound to the contacting (inner) surface of the 30S and the second aminoacyl-tRNA (lys-tRNA) to the contacting surface of the 50S subunit. L P, and A represent the initiation, peptidyl, and aminoacyl binding sites. When the ribosome locks, the A and P sites overlap [2]
9 by Springer-Verlag 1976
347
at the interface between the two ribosomal subunits as depicted in Figure 2 for prokaryotic ribosomes.
cleotides such as A U G A A A A A A . . . A A A ( A U G A , ) , beginning with an initiatior codon A U G at the 5' end, yielded Met-Lys-Lys...Lys oligopeptides with methionine (Met) at the amino-terminal end, the remaining amino acid residues being all lysine (Lys) residues. Thus, a small a m o u n t of [t4C]methionine and a larger a m o u n t of [~4C]lysine was incorporated into acid-insoluble products. When similar oligonucleotides but beginning with a non-initiator codon (e.g., the glycine codon, G G U ) were used ( G G U A , ) their translation required a higher Mg 2+ concentration and, in any case, the non-initiating codon was not translated; the resulting peptides contained only [14C]lysine [4]. As seen in Figure 3, the addition of initiation factors IF-1 and IF-2, partially purified f r o m the ribosomal wash, was essential for translation of oligonucleotides beginning with an A U G codon at the 5' end, whether at 14 m M or 18 m M Mg 2+. On the other hand, oligonucleotides beginning with G G U which were not translated at 1 4 r a M Mg 2§ with or without factors, were translated at 18 m M Mg 2 + in the absence of added factors. Table 1 summarizes some of the early results of our laboratory [3, 5]. At first, we washed the ribosomes with 0.5 M NH4C1 and, utilizing the f M e t - t R N A r ribosomal binding assay referred to below, isolated and partially purified two protein factors, IF-1 and IF-2. These factors (Table 1, experiment 1) were not required for translation of poly(A) and their addition produced n.o stimulation. However, translation of natural messengers, such as the R N A s of the coilphages MS2 and Q/~, required addition of the factors. It was later found that, when the ribosomes were washed with 1.0 M NH4C1, one other factor, IF-3, was required for natural messenger translation (Table 1, experiment 2). Ribosomes can bind fMet-tRNAf, the initiator aminoacyl-tRNA, to form an initiation complex upon incubation in the presence of the trinucleoside-diphosphate A p U p G ( A U G ) as messenger. Ribosomal binding of f[14C]Met-tRNA is readily measured because the ribosome-bound, but not the free c o m p o u n d is retained by nitrocellulose filters following incubation
Initiation Factors: Nature and Function
The polypeptide chain initiation factors were discovered in 1965 through the use of E s c h e r i c h i a coil ribosomes that had been washed with concentrated salt solutions. These ribosomes, while translating synthetic messengers, such as poly(U) or poly(A), were unable to translate natural messengers unless the ribosomal wash was added back. Washed ribosomes were also able to translate oligonucleotide messengers that had non-initiating codons at the 5' terminus, but those having an initiator codon A U G in this position, required addition of the ribosomal wash for translation [3]. This finding was essential for establishing that the factors present in the ribosomal wash were in fact concerned with initation. Translation of oligonu-
1400 _~1200
-
tO
+Z -F
[']kysine
I
E
=
F=initiotion foctors Methionine
800-
O n
8
I 600
I
-o
'~ 400
E
o
200
0
-F l AUGA18GGUA2~AUGAIBGGUA2~ 14ml Mg2+ 18mM Mg2+
Fig. 3. Effect of initiation factors and Mg2+ on the translation of the oligoribonucle0tides AUGAn and GGUAn. A nuclease-low cell-free system,consisting of NH4Cl-washed E. coli Q13 ribosomes and LactobacilIus arabinosus high-speed supernatant was used. Incubation 40 min at 37 ~ +F, addition of initiation factors (IF-1 and IF-2); - F , no factor additions (prepared from Table 1 of [4])
Table 1. Effect of initiation on translation of natural messengers by NH4Cl-washed E. coli ribosomes (from [3, 5]). The translation system consisted of washed ribosomes and high-speed supernatant from E. coli Q13. The Mg2+ concentration was 18 mM for poly (A) translation and 14 mM in all other cases. Incubation was at 37 ~ for 40 min in experiment 1 and 20 min in experiment2 Exp. No.
Washingof ribosomes
mRNA
0,5 M NH4CI
Poly (A)
Q~ RNA
348
Amino acid incorporation [pmotes/sample] Lysine
MS2 RNA
1.0 M NH4C1
Factor additions
MS2 RNA
None IF-1 +IF-2 None IF- i + IF-2 None IF-I +IF-2 None Ribosomal wash IF-1 +IF-2 IF-3 IF-1 +IF-2+IF-3
4,898 4,518 64 606 332 1,200 20 980 45 355 1,082
Leucine
Methionine Histidine
68 767
15 155
Naturwissenschaften 63, 347-355 (1976)
10 92
9 by Springer-Verlag 1976
r-~
A.
u m
o 12 E
,=,
B.
fMet - tRNAf + I F - I + TF-2
"o
Q 1 0
= I0 :=
E
IF-I +IF-2
o "o
,Z
c
z
8
o .o
r,-
L :S
6
z er
,q, im
I
I
4
IF-2
7
only
2 I~"
Met-tRNAf
I
with I F - I
+TF-2
0 2
0
4 MINUTES
6
8
O 0
I0
2
4 6 MINUTES
8
Fig. 4. (A) Effect of IF-1 +IF-2 on the AUG-dependent binding of formylated and non-formylated fMet-tRNAf to E. coli ribosomes (from [4]). (B) Effect of IF-1 and IF-2, singly and in combination, on the AUG-dependent ribosomal binding of fMet-tRNAf (from [6]). Reactions were conducted at 5 mM Mg z+ and 25 ~
Table 2. AUG-dependent binding of fMet-tRNA r with ribosomal subunits at 0 ~ and 25 ~ (from [71). The ribosomal binding of t~C]Met-tRNAf was determined by the nitrocellulose filter procedure after incubation for 15 min in the presence of 3.5 mM Mg z§ 0.3 m M GTP, and AUG as messenger, at the indicated temperature Temp. [~
0
Subunits
30S 30S + 50S
25
30S 30S + 50S
Factor additions [ g g ] IF-2
IF- 1
f[14C]MettRNAf bound [moles]
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
None 1.0 None 1.0 None 1.0 None 1.0
1.4 1.7 4.0 8.0 0.4 1.9 0.7 4.8
Stimulation by IF-1
x 1.2 x2.0 x4.7 x6.8
in the presence of Mg 2+. Figure 4A shows the requirement of IF-1 and IF-2 for binding at 3 m M Mg 2 +. Under these conditions, non-formylated MettRNAf is not bound whether in the presence or absence of factors. In Figure 4B it may be seen that both IF-1 and IF-2 were needed for maximal ribosomal binding of f[~4C]Met-tRNAf and that dependence on IF-2 was complete. However, although there was a marked decrease in binding in the absence of IF-l, the requirement for this factor was not absolute. Is IF-1 an Initiation Factor?
When the binding is carried out a low temperature the dependence on IF-1 is markedly decreased. Table 2 shows that, when the temperature of incubation was dropped to 0 ~ there was good binding in the presence of IF-2 alone, whether with 30S or with 30S+50S subunits. At this temperature IF-1 Naturwissenschaften 63, 347-355 (1976)
produced only a moderate increase (up to two-fold) in binding. However, at 25 ~ the stimulation by IF-1 was five to seven-fold. These results suggest that IF-1 merely increased the stability of the initiation complex which is lower at the higher temperature [7]. Thus, IF-1 might be a component protein of the 30S initiation site which, unlike other ribosomal proteins, is readily extracted from E. coli ribosomes by salt washing. The view that IF-1 may be a ribosomal protein rather than an initiation factor is strengthened by the finding of Leffler and Szer in our laboratory [8] that no IF-Mike proteins were obtained by salt-washing of Caulobacter crescentus ribosomes. Moreover, in marked contrast to E. coli ribosomes, the AUG-directed, IF-2-dependent binding of fMet-tRNAe to C. crescentus ribosomes was as good at 24 ~ as at 0 ~ (Table 3, experiment A). It may also be seen (Table 3, experiment B) that addition of E. coli IF-1 caused no stimulation of initiation complex formation with C. crescentus ribosomes and C. crescentus phage Cb5 RNA at 37 ~ whereas the factor markedly stimulated complex formation by E. coli ribosomes with the E. coli-specific MS2 phage RNA. Recycling of IF-2, i.e., the alternating association and dissociation of the factor to and from ribosomes during protein synthesis (see below) may depend on the integrity of the 30S initiation site. This might explain why IF-1 is required for IF-2 recycling [9]. IF-1 has been isolated in crystalline form [10]; it is a small basic protein (molecular weight 9,400). It contains all of the common 20 amino acids with only one residue each of histidine, proline, cysteine, methionine, and tryptophan, and has alanine and lysine, respectively, at the amino and carboxy terminal ends. The amino acid composition of IF-1 is similar to that of the majority of ribosomal proteins which, although differing widely in molecular weight, are remarkably similar in amino acid composition.
9 by Springer-Verlag 1976
349
IF-2 is indispensable for formation of the 30S initiation complex and appears to recognize and promote the ribosomal binding of only one species of naturally occurring aminoacyl-tRNA, namely, the initiator fMet-tRNAf. In this regard, its function is similar to that of the chain elongation factor Tu which does not recognize the initiator but does recognize and promote the binding of all of the other aminoacyl tRNAs to the 70S ribosomal complex. IF-2 functions catalytically [12]. One molecule of IF-2 can promote the binding of several molecules of fMet-tRNA to ribosomes. Since IF-2 itself is bound to the 30S ribosomal subunit when the 30S complex is formed, it must be eventually released to become available for new rounds of initiation. This happens when the 50S subunit joins the 30S complex to form the 70S initiation complex (Fig. 1, step 2). This recycling of IF-2 requires GTP. No recycling is possible when the analog 5'-guanylylmethylene diphosphonate (GMPPCP), that cannot be hydrolyzed, is substituted for GTP, or when the 70S initiation complex is formed with
Table 3. Initiation factor requirements for formation on initiation complex with Caulobacter crescentus and Escherichia coli ribosomes (from [8]). Binding measured as in Table 2. A. Incubation was for 15 min at 24 ~ and 45 min at 0 ~ B. Incubation was for 10 min at 37 ~ A (Template, AUG) Source of washed ribosomes
pmoles of [l'~C]Met-tRNAfbound in the presence of C. crescentus IF-2
C. crescentus
ribosomal wash
C. crescentus E. coli E. coli+ IF-1
at0~
at 24~
at0~
at 24~
3.9 3.0
3.8 0.7 4.0
3.8 3.5
3.6 1.0 4.5
B (Template, natural mRNA) Source of washed ribosomes
mRNA
C. crescentus
Phage Cb5
IF-1
(C.crescentus)
(E. coli) tRNAf
f[a*c]Metbound [pmoles]
Phage MS2
E. coli
IF-2
"70 S 50 S
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Fraction number Fig. 5. Interaction of 3aP-labelIed tF-2 with ribosomes. A 30S initiation complex was first formed with f[3H]Met-tRNAf ( e - - e ) and [32p]IF-2 ( o - - o), in the presence of either GMPPCP or GTP, by incubation for a further 5 min the samples were analyzed by sucrose density gradient centrifugation. (A) with GMPPCP and (B) with GTP (from [11])
0.8 --
06
-
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g)
0"4
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--
E. coli IF-2 has been purified to near homogeneity
in several laboratories [11]. The factor appears to exist in a t least two forms, IF-2a and IF-2b of molecular weights around 80,000 and 90,000, respectively. IF;2 is sensitive to sulfhydryl binding reagents such as p-mercuribenzoate or N-ethylmaleimide. 350
6. DF activity of IF-3. E. coli ribosomes with a high proportion of 70S couples were incubated for 20 min at 37 ~ (6.5 mM Mg 2 +) with n o factors (A), IF:I (B), or IF-3 (C), and the distribution of 30S, 50S, and 70S ribosomes was analyzed by sucrose gradient centrifngation (from [16]) Fig.
Naturwissenschaften 63, 347--355 (1976)
9 by Springer-Verlag 1976
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