Eur. J. Biochem. 44, 123-130 (1974)

Synthesis of Individual Ribosomal Proteins during a Nutritional S hift-Up Graham CARPENTER and Bruce H. SELLS Laboratories of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland

(Received January 16, 1974)

Although individual ribosomal proteins are synthesized at the same rate during exponential growth, during a nutritional shift-up, they are synthesized a t different rates. Proteins S6, SlO, S14,S20 and L2, L3, L4 and L13 are synthesized to the greatest extent. Proteins 52, 53, 54, S13, S17 and L7, L8, L9, L25, L29 and L30 are synthesized a t the lowest rate. Although the average rate of synthesis of 50-5 protein is greater than that for 30-5 protein during a shift-up, the data on rates of synthesis of individual proteins do not support such a distinction for each individual ribosomal protein.

The extent of increased synthesis for the individual ribosomal proteins of the 30-5 subunits correlated well with the assembly order of these proteins. This correlation is not as striking for the 50-S subunit.

Ribosome particles are composed of approxi- mately 50 distinct proteins. Over the past several years studies initiated by Nomura's group [i] have demonstrated a sequence of assembly in vitro of groups of ribosomal proteins in the formation of the individual subunits. Studies from our laboratory [2,3] have also indicated a sequence of assembly of groups of ribosomal proteins during ribosome forma- tion in vivo. More recent data [4,5] have provided additional support for this belief. How the synthesis of ribosomal proteins and the formation of ribosomal particles is controlled has not been clearly established. The investigations presented in this manuscript were designed to determine whether synthesia of ribosomal protein is controlled collectively or whether each individual ribosomal protein is regulated in- dependently. Advantage was taken of the nutritional shift-up system in which an exaggerated rate of synthesis of ribosomal proteins occurs [4,5]. At various times following shift-up, the rate of synthesis of individual ribosomal proteins was determined. The results indicate that early during shift-up the individual ribosomal proteins may be arranged into groups according to their rates of synthesis. In such an arrangement, there appears to be four groups of proteins in the 50-S subunit which are synthesized at different rates. A similar grouping can be demon- strated for the 30-5 subunit.

MATERIALS AND METHODS Bacteria and Culture Conditions

Escherichia coli H128 was grown at 30 "C in Cohen's salts medium [6] supplemented either with

acetate or with glucose, amino acids and nucleosides as previously described 1171.

Isotopes

~-[4,5-aH,]Lysine (spec. act. 55 Cilmmol) and ~-[U-14C]lysine (spec. act. 310 mCi/mmol) were ob- tained froin New England Nuclear.

Labelling of Ribosomal Proteins

To measure the rate of synthesis of individual ribosomal proteins under different conditions, the following procedure was used. Cells were incubated with [14C]lysine (0.10 pCi/ml and 47 ng/ml) for 1 min. Incorporation of the isotope was stopped by the addi- tion of an excess of unlabelled lysine (1.5 mglml), and the cells were incubated for an additional 45 min to allow labelled ribosomal protein to be assembled into mature subunits. The amount of 14C-labelled protein present in the ribosomes, there- fore, was an index of the amount of protein synthesiz- ed during a 1-min interval. The cells were then poured over crushed ice and an aliquot of cells, previously in- cubated with [3H]lysine (2.0 pCi/ml and 10 mg/ml) for 5 to 6 generations during exponential growth in the acetate medium, was added. Incubation with [aH]lysine permitted labelling of ribosomal proteins in proportion to lysine contents. The mixed sH and 14C-labelled cells were then centrifuged, washed once with buffer (10 mM Tris-C1 pH 7.8, 10 mM MgCl and 1 mM dithiothreitol) and stored as a pellet at -20 "C.

Eur. J. Biochem. 44 (1974)

124 Synthesis of Ribosomal Proteins during a Nutritional Shift-Up

Isolation of Ribosomal Proteins The cells were resuspended, disrupted in a French

pressure cell and NH4C1-washed ribosomal subunits prepared as previously described [S]. To each sub- unit preparation approximately 7 mg of unlabelled 30-S or 50-S subunits were addcd. These carrier sub- units were prepared as described by Kurland [9] and Staehlin and Maglott [ 101. Ribosomal protein was extracted with acetic acid as previously described [ll] and dialyzed overnight against the one-dimen- sion electrode buffer described by Kaltschmidt and Wittmann [12]. The protein samples were concen- trated to a small volume (approximately 0.3 ml) with dry sucrose [ I l l . The concentrated protein samples were then dialyzed overnight against the buffer of Kaltschmidt and Wittmann [12].

Separation of Ribosomal Protein Individual ribosomal proteins were separated in

the two-dimensional polyacrylamide gel system described by Kaltschmidt and Wittmann [12]. After electrophoresis, the proteins were stained with analine blue black for 20 min. The gels were destained by rinsing with a solution containing 70/, acetic 3O/, ethanol for 24 h followed by rinsing with 101, acetic acid.

ikfeasuvement of Radioactivity The discs of gel containing the stained ribosomal

proteins were removed from the slab gel with a cork bore, broken into small pieces and placed in a scintil- lation vial with 0.15 ml water and 2.0 ml Protosol. The vials were tightly capped and incubated at 40 "C for 18 h. 10 ml scintillation fluid (0.40/, omni- fluor in toluene) was added to each vial and the vials were counted in a Beckman LS-255 liquid scintilla- tion counter. The 14C and 3H counts were corrected for isotope spillover and background. Quenching was monitored with the external standard.

Isolation of Elongation Factors Elongation factors were isolated by DEAE-cellu-

lose chroniatography and disc gel electrophoresis as will be described elsewhere.

RESULTS Synthesis of Ribosomal Proteins during Exponential Growth

These initial experiments were designed to establish the relative rates of synthesis of individual ribosomal proteins in the course of a 1-niin pulse during exponential growth. These rates were measur- ed during two different conditions of growth: (a) in acetate minimal medium (mass doubling time

160 min) and (b) in enrichment medium (mass dou- bling time 60 min). To define the rate of ribosomal protein synthesis in a particular medium, a suspen- sion of E. coli growing exponentially in that medium was incubated with [14C]lysine for 1 min. Incorpora- tion was arrested by the addition of an excess of unlabelled lysine and the incubation continued for an additional 45 rnin to allow for the assembly of the I4C-labelled protein into mature ribosomal sub- units. These cells were then mixed with cells with had previously been incubated with [SHIlysine in an identical medium for six generations. Following combination of the 3H and 14C-labelled cells, the ribosomal proteins were isolated and separated into individual species by two-dimensional polyacryl- amide gel electrophoresis. The 14C/3H ratios of the individual ribosomal proteins, obtained from cells grown in the enriched medium and in the acetate medium (minimal medium), are presented in Tables 1 and 2, respectively. The normalized values of the 14C/3H ratios are also presented in these tables and are an expression of the relative rates of production of each protein during a I-min pulse. The data in Table 1 show that during exponential growth of E. coli in the enriched medium, all the ribosomal proteins are synthesized a t approximately equal rates. The data in Table2 show similar results for growth in the acetate medium. It should be noted, however, that in the acetate medium proteins S6, S20 and L33 have significantly higher normalized ratios (30 -55O/,) than do other ribosomal proteins, indicating that they are synthesized a t higher rates. This observation will be considered in more detail in the Discussion.

Synthesis at 20 M i n after a Shift-Up The following experiments were performed to

determine the rate of synthesis of individual ribo- somal proteins at various times during the growth rate transition following nutritional shift-up. During a nutritional shift-up, a preferential increase in the rate of total ribosomal protein synthesis occurs immediately after the cells are exposed to an enriched nutritional environment [8, 131. We have demon- strated that a t 20 min following a shift-up, the rates of synthesis of 50-5 subunit proteins and 30-5 sub- unit proteins reach a maximum and are increased 6.0-fold and 3.0-fold, respectively, (unpublished observations). The synthesis of individual ribosomal proteins was examined to determine whether the rate of synthesis of all ribosomal proteins is increased equally.

A suspension of E. coli growing exponentially in acetate medium was incubated with [3H]lysine for five generations. The cells were removed from the acetate medium and transferred to the enriched medium as previously described [7]. After 20 min of

Eur. J. Biochem. 44 (1974)

G. Carpenter and B. H. Sells 125

Table 1. Synthesis of individual ribosomal proteins during balanced growth in enriched medium

Pro- 14C/3H Normal- Pro- 14C/3H Normal- tein ratio ized tein ratio ized

ratio ratio

s1 s 2 s3 84 55 S6 57 S8 s 9 810 S l l s12 513 S14 815 S16 517 518 s19 s20 521

0.120 0.147 0.121 0.131 0.131 0.146 0.135 0.130 0.126 0.147 0.150 0.138 0.129 0.133 0.131 0.146 0.147 0.149 0.142 0.132 -

0.86 1.05 0.87 0.87 0.94 1.05 0.97 0.94 0.90 1.05 1.08 0.99 0.93 0.96 0.94 1.05 1.05 1.07 1.02 0.95 -

L1 L2 2 2 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 LS9 L20 L21 L22 L23 L24 L25 L26 L27 L28 L29 ~ 3 0 -~ L31

Elonga- L32 tion 0.145 1.04 L33 factors L34

0.135 0.149 0.139 0.151 0.150 0.139 0.156 0.123 0.136 0.140 0.141 0.128 0.128 0.125 0.127 0.138 0.135 0.126 0.132

0.144 0.136 0.139 0.139 0.140 0.160 0.150 0.151 0.131 0.144 0.140 0.165 0.136 0.144

-

0.97 1.07 1.00 1.09 1.08 1.00 1.12 0.88 0.98 1.01 1.01 0.92 0.92 0.90 0.91 0.99 0.97 0.91 0.95

1.04 0.98 1.00 1.00 1.01 1.15 1.08 1.09 0.94 1.04 1.01 1.19 0.98 1.04

-

growth in the new medium, the cells were labelled with [14C]lysine as described in Materials and Methods and a t 21 min an excess of unlabelled lysine was added. Incubation was continued for an additional 45 min, the cells collected and the individual ribo- somal proteins isolated as described in Materials and Methods. The 14C/aH ratios and their normalized values for each ribosomal protein are presented in Table3. In this experiment, the increased rate of synthesis of total 30-S proteins and total 50-S pro- teins was identical to that which we have found previously (unpublished observations). An examina- tion of the W/3H values and normalized ratios in Table 3 shows that at 20 min following a shift-up, nearly ell of the individual ribosomal proteins are synthesized a t equivalent rates.

Ribosomal Protein Synthesis Early during Nutritional Shift- U p

The results in the previous section indicated that a t the time when total ribosomal protein formation had reached its maximum rate, the individual ribo-

s1 s 2 s 3 54 s 5 S6 s 7 S8 s 9 s10 S l l 512 S13 514 515 S16 S17 S18 s19 520 521

0.053 0.041 0.042 0.048 0.050 0.073 0.048 0.043

0.043 0.051 0.048

0.049 0.053 0.046 0.043 0.039 0.049 0.063

-

-

-

1.13 0.87 0.89 1.02 1.06 1.55 1.02 0.91

0.91 1.08 1.02

1.04 1.13 0.98 0.91 0.83 1.04 1.34

-

-

Table 2. Synthesis of individual ribosomal protein during balanced growth in acetate minimal medium

Pro- 14C/3H Normal- Pro- 14C/3H Normal- tein ratio ized tein ratio ized

ratio ratio

,

soma1 proteins were all synthesized at essentially the same rate. The following experiment was per- formed, therefore, to determine whether the initial exposure of bacterial cells to an enriched nutritional environment resulted in a simultaneous increase in the rate of synthesis of each individual ribosomal protein. A suspension of E. coli growing exponentially was divided equally into four flasks. When the cells reached the mid-log phase of growth, a concentrated mixture of nutrients (glucose, amino acids and nucleosides) was added to cach flask. The final con- centration of each added nutrient was identical to that used to prepare enriched medium in previous experiments. At 0, 2, 4 and 6 min following the addi- tion of nutrients, one of the flasks was selected and [14C]lysine was added. One minute later the incorpora- tion of isotope was stopped by the addition of an excess of unlabelled lysine, and incubation continued for an additional 45 min to allow the labelled ribo- somal proteins to be assembled into mature subunits. To each of the 14C-labelled samples, equal volumes of cells previously labelled with [SHIlysine during six generations of growth in acetate medium were

Elonga- tion 0.050 1.06

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 E l 5 LS6 1,17 L18 Ll9 L20 L21 L22 L23 L24 L25 L26 L27 L28 L29

L31 L32 L33

L30

factors L34

0.045 0.046 0.043 0.042 0.044 0.044 0.042 0.048 0.043 0.042 0.047 0.047 0.043 0.046 0.042 0.046 0.047 0.039 0.044 0.045 0.048 0.044 0.042 0.044 0.041 0.038 0.050 0.035 0.046 0.047 0.046 0.051 0.071 0.047

0.96 0.98 0.91 0.89 0.94 0.94 0.89 1.02 0.91 0.89 1 .oo 1.00 0.91 0.98 0.89 0.98 1.00 0.83 0.94 0.96 1.02 0.94 0.89 0.94 0.87 0.81 1.06 0.81 0.98 1.00 0.98 1.08 1.51 1 .oo

Eur. J. Biochem. 44 (1974)

126 Synthesis of Ribosomal Proteins during a Nutritional Shift-up

Table 3. Synthesis of individual ribosomal proteins and elonga- tion factors at 20 min following a nutritional shift-up

Pro- 14C/SH Normal- Pro- 14C/SH Normal- bin ratio ized tein ratio ized

ratio ratio

s1 52 53 54 s5 S6 s7 58 s9 s10 Sll 512 S13 514 515 S16 517 S18 s19 520 521

- 0.086 0.107 0.115 0.120 0.144 0.111 0.137 0.104 0.102 0.113 0.108 0.113 0.122 0.132 0.103 0.103 0.131 0.113 0.113 0.088

- 0.83 1.04 1.12 1.16 1.40 1.08 1.33 1.01 0.99 1.19 1.05 1.10 1.18 1.22 1.00 1 .oo 1.27 1.10 1.10 0.85

rAi L2 L3 L4 L5 LG L7 L8 L9 L10 L11 L12 L13 L14 215 L16 L17 L18 Ll9 L20 L21 L22 L23 L24 L25 L26 L27 L28 L29 L30

Elonga- L31 tion 0.043 0.42 L32 factors L33

L34

0.094 0.108 0.095 0.096 0.117 0.110 0.082 0.086 0.105 0.097 0.106 0.166 0.094 0.095 0.094 0.080 0.096 0.104 0.087

0.067 0.088 0.103 0.105 0.093 0.082 0.102 0.082 0.106 0.096 0.107 0.129 0.094 0.111

-

0.91 1.05 0.92 0.93 1.14 1.07 0.80 0.83 1.02 0.94 1.03 1.61 0.91 0.92 0.91 0.78 0.93 1.01 0.84

0.65 0.85 1.00 1.02 0.90 0.80 0.99 0.80 1.03 0.93 1.04 1.25 0.91 1.08

-

added. The double-labelled samples were then centri- fuged and the individual ribosomal proteins isolated as described in Materials and Methods. In this experi- ment, the rate of synthesis of 30-S proteins and 50-S proteins increased by 57 and 96O/,, respectively. The 14C/3H ratios obtained for the individual ribo- somal proteins, synthesized a t 0, 2, 4 and 6 min following shift-up, are for 30-5 and 50-S subunits as shown in Tables4 and 5 , respectively. The data in these tables show that not all ribosomal proteins are synthesized a t the same rate during a nutritional shift-up. There is an approximate %fold difference between the highest and lowest rates of synthesis as indicated by the range of l4CI3H ratios of the individual ribosomal proteins. These results show that a simultaneous increase in the rate of synthesis of all the ribosomal proteins does not occur; rather, some proteins are synthesized a t a high rate relative to the others, e.g. 56, SIO, 514, S18, 520 andL2, L3, L4 and L18. A number of ribosomal proteins, e.g. S2, S3, 54, S13, 517 and L7, L8, L9, L25, L29 and L30 are synthesized at a relatively low rate. A com- parison of the ratios obtained for 3 0 3 proteins with

Table 4. Synthesis of individual ribosomal proteins of the 30-5 subunit during first 5 min of shif t-up

Time of labelling

0-1 min 2-3 min 4-5 min

Protein l*C/SH Protein 14C/3H Protein l*CrH ratio ratio ratio

s20 s10 S6 514 s11 S16 518 s7 512 s 9 515 s2 S8 S13 s1 54 s5 s19 53 S17

0.178 0.168 0.164 0.147 0.136 0.136 0.136 0.134 0.133 0.128 0.124 0.117 0.116 0.116 0.115 0.112 0.105 0.101 0.099 0.094

520 S6 s10 S14 S18 S16 s7 515 S8 s 9 512 s19 s11 s5 s4 513 52 s 3 S17

0.205 0.196 0.176 0.172 0.150 0.145 0.137 0.136 0.135 0.133 0.131 0.129 0.117 0.113 0.106 0.094 0.087 0.083 0.078

s10 s20 S15 S6 S14 S18 S16 88 s9 s19 s7 s5 512 93 54 S11 517 S13 s2

0.282 0.273 0.267 0.258 0.247 0.219 0.216 0.208 0.207 0.204 0.188 0.184 0.179 0.166 0.144 0.142 0.137 0.130 0.099

the ratios obtained for 50-S proteins indicates that while the total 50-5 proteins are synthesized a t a slightly higher rate than total 30-S proteins, aa previously reported [8], several 3 0 3 proteins are synthesized a t higher rates than many of the 50-5 proteins. The 14C/SH ratios for proteins S6, SIO, S14, S18 and S20 are considerably higher than the ratios for 50-S proteins L7, L8, L9, L25, L29 and L30. It should be noted, also, that the rate of synthesis of these 30-5 proteins is not as high as the 50-5 proteins L2, L3, L4 and L18. Therefore, it does not appear that the synthesis of 30-5 proteins and 50-S proteins are totally separated processes.

Synthesis of Elongation Factors The rate of synthesis of elongation factor was

measured to determine how the synthesis of these proteins might be related to the synthesis of indi- vidual ribosomal proteins. Nomura and Engbaek [la] have suggested that these proteins may be part of the same transcriptional unit as the ribosomal proteins. The rates of synthesis of the elongation factors were measured during the same conditions of balanced growth and nutritional shift-up as the ribosomal proteins. The labelling conditions for these experiments was identical to those described for the studies of ribosomal proteins previously outlined. The double-labelled elongation factors were obtained from the soluble phase of the cell-free preparation following removal of the ribosomes. The data (l4CISH ratios) in Tables I and 2 show that the

Eur. J. Biochem. 44 (1974)

G. Carpenter and B. H. Sells 127

Table 5. Synthesis of individual ribosoml proteins of the 50-8 subunit during the first 7 min of a shift-up

Time of labelling ~~

0-1 min 2-3 min 4-5 min 6-7 min

Protein 14C/3H ratio Protein l4C/8H ratio Protein 14C/3H ratio Protein I4C/3H ratio

L12 L13 L11 L17 L18 L14 L27 L19 L21 L15 L33 L16 L26 L28 L3 L5 L25 L32 L31 L4 L24 L1 L10 L23 L2 L6 L34 L9 L30 L29 L22 L8 L7

0.400 0.319 0.317 0.283 0.232 0.231 0.196 0.196 0.195 0.188 0.188 0.174 0.167 0.162 0.154 0.154 0.151 0.148 0.147 0.144 0.143 0.130 0.127 0.124 0.123 0.121 0.110 0.108 0.102 0.099 0.098 0.091 0.085

L17 L4 L3 L18 L27 L14 L33 L12 L13 L11 L23 L1 L10 L21 L5 L26 L31 L32 L6 L2 L19 L24 L28 L9 L25 L15 L8 L34 L7 L22 L16 L29 L30

0.227 0.224 0.221 0.220 0.216 0.207 0.207 0.204 0.203 0.197 0.193 0.192 0.192 0.190 0.185 0.181 0.170 0.168 0.166 0.164 0.152 0.152 0.149 0.132 0.129 0.127 0.125 0.123 0.108 0.105 0.099 0.094 0.091

L3 L18 L4 L11 L1 L13 L2 L33 L17 L6 L22 L27 L5 L12 L31 L14 L32 L21 L10 L24 L19 L23 L28 L15 L26 L16 L25 L9 L29 L8 L30 L7

0.320 0.319 0.314 0.299 0.296 0.283 0.273 0.265 0.262 0.258 0.254 0.253 0.249 0.242 0.241 0.226 0.225 0.222 0.221 0.215 0.210 0.209 0.201 0.198 0.187 0.182 0.181 0.177 0.171 0.165 0.138 0.134

~ -

L12 L18 L4 L3 L32 L2 L13 L14 L29 L33 L31 L32 L5 L6 L17 L11 L1 L19 L24 L27 L15 L16 L26 L21 L34 L10 L22 L9 L28 L8 L30 L25 L7

0.416 0.355 0.321 0.312 0.311 0.301 0.298 0.269 0.262 0.260 0.258 0.255 0.253 0.249 0.249 0.248 0.246 0.243 0.243 0.242 0.234 0.227 0.219 0.216 0.213 0.193 0.191 0.183 0.180 0.175 0.174 0.160 0.150

elongation factors are synthesized a t the same rate as individual ribosomal proteins during balanced growth a t different growth rates. The results in Table 3, however, show that the elongation factors are synthesized at a much lower (approximately 60 rate than individual ribosomal protein during a shift-up. This finding substantiates our previous unpublished conclusion that the control of elongation factor synthesis is not identical to that of the ribo- somal proteins.

DISCUSSION The present studies were designed to determine

whether production of each individual ribosomal protein is under one common control or whether each is under separate control. During the course of these studies, several observations were made which should be noted.

Synthesis of Individual Ribosomal Proteins during Exponential Growth

Under the conditions of balanced growth in enriched medium, all 60-5 and 30-S ribosomal proteins are synthesized a t the same rate during the 1-min pulse with the labelled amino acid. During growth in the acetate medium, although most ribo- somal proteins are synthesized at essentially the same rate, three exceptions have been observed (86,520 and L33). The interpretation of this observa- tion is complicated by the fact that these proteins are fractional ribosomal proteins. Deusser [ 151 has shown that S20 and L33 are present in the amount of 0,6mol/mol 30-S subunits or 50-S subunits, respectively, while approximately 0.1 -2.5 mol S6 is found per mol 30-S subunit. These fractional proteins may exist in non-ribosomal pools in the cell and may not partition between the ribosomes and the non-ribosomal pools in the same manner in cells

Eur. J. Biochem. 44 (1974)

128 Synthesis of Ribosomal Proteins during a Nutritional Shift-Up

labelled for 1 min with [14C]lysine and in cells labelled for six generations with [3H]lysine. Because of the lack of information about these fractional ribosomal proteins, i t is difficult to conclude that the 14C/3H ratios necessarily indicate a high level of synthesis. Since none of these fractional proteins has high or low ratios in cells grown in enriched medium (Table 1) the abnormal ratios observed during growth in the acetate medium may reflect events which occur only a t low rates of growth.

Synthesis of Individual Ribosomal Proteins 20 M i n after Nutritional Shift- U p

Following nutritional shift-up, the rate of total ribosomal protein synthesis increases to a maximum approximately 20min after the cells have been exposed to enriched medium. Although most indi- vidual ribosomal proteins are synthesized a t equal rates 20 min following shift-up, several proteins have 14C/3H values which are different from the 14C/3H ratios of the majority. Proteins L7 and L12 fall into this category. The ratios observed for proteins L7 and L12, however, cannot be considered as indicative of rates of synthesis. Proteins L7 and LIZ differ only by the presence of an acetyl group [i6] and have the same primary structure [i7]. The relative amounts of L7 and Li2 furthermore depend upon the growth rates [13,18]. Since the proteins L7, LIZ were labelled with 3H in acetate medium and labelled with 14C in enriched medium, the ratio of 14C/3H reflects two different processes : (a) synthesis of L7 and L12 and (b) post-translational changes in the ratio of L7 and L12 caused by acetylation of these proteins. There are no data, available to assess the extent to which acetylation occurs during growth in acetate medium which supports a generation time of 160min.

It should be also noted that proteins S2, 56, S16, 818, L26, L28 and L32 which have normalized ratios either 20°/, higher or lower than the average value are fractional proteins [13,19]. Interpretation of the ratios obtained for these proteins may be complicated by factors such as partitioning of these proteins between ribosomes and cell cytoplasm when the cells are labelled for different lengths of time and in different media, as discussed above. Deusser [i5] has reported that the stoichiometry of proteins S6 and L16 varies according to the growth rate of the cells.

Synthesis of Individual Ribosomal Proteins Early in Shift-Up

These studies indicate that early during shift-up, the rates of individual ribosomal proteins are syn- thesized a t several different rates. The individual ribosomal proteins of the 30-5 subunit can be arrang- ed into four groups as follows:

Group I S - S20, SiO, S6, S14 Group I1 S - S16, S18, Si5, S8, S9, 57 Group I11 S - Sl9, S12, 55, Sll , Sl Group IV S - S4, Si3, S17, 53, 52.

I n constructing these groups, only the data ob- tained during the 2-3-niin and 4-5-min pulses have been employed. Data from 0-1-min pulse were not used since during this period it was assumed that the ribosomal proteins synthesized would be influenced by the translation of messenger RNA’s transcribed just prior to shift-up.

The proteins in group1 are synthesized a t the highest rate and those in groupIV a t the lowest rate during the shift-up. The protein listed first in each group is judged to have the highest rate of synthesis in that group and the last protein the lowest rate of synthesis. Since the groups cannot be un- ambiguously defined, the placing of some proteins is arbitrary. For example, protein S7 could be placed first in group111 instead of last in groupI1. The placement of protein S1 is based upon only one measurement and cannot be considered secure. Protein 521 was not detected in these studies. Pro- teins S1 and 521 are fractional proteins which are present on only loo/, of the ribosomes in a cell [15,i9]. This fact may account for the difficulty of consistent- ly locating these proteins in the two-dimensional gel system.

The arrangement of the 50-5 proteins into groups according to their rates of synthesis was more com- plex than that for the 30-S proteins. A grouping of the 50-S proteins according to their relative rate of synthesis as described previously for the 30-5 pro- teins is as follows:

Group I L - L18, L4, L3, L13 Group I1 L - L14, L33, L5, L31, L32 Group I11 L - L6, L19, L24, L15, Li6, L26, L28,L34 Group IV L - L25, L29, L9, L8, L30, L7.

The grouping of nine proteins (Li, L10, L l l , L12, Li7, L21, L22, L23 and L27) could not be firmly established and their location within a group was not possible. Protein L20 was not located on the two-dimensional gel system used to separate the ribosomal proteins. Weber [19] has reported a similar difficulty in the detection of L20.

Genetic Location of Cistrons for Ribosomal Proteins

If the order of increase in the rates of synthesis of the individual ribosomal proteins is postulated to reflect the sequence of transcription of the cistrons for these proteins, models of ribosomal protein genes can be constructed which would account for the observed results. The order of synthesis suggests that the group I proteins of the 50-5 subunits which

Eur. J. Biochem. 44 (1974)

G. Carpenter and B. H. Sells 129

have the highest rates of synthesis are located a t the promoter proximal end of a ribosomal protein oper- on(s). The groupIV proteins of the 30-5 subunit which have the lowest rates of synthesis would be located at the promoter distal end of an operon(s). Nomura and Engbaek [14] have presented genetic data which suggest that ribosomal proteins determin- ed by the ery, spcA, strA and fus loci are part of one transcriptional unit and that the order of transcrip- tion is ery, spcA, strA and fus. Our data indicate that the ery protein, L4 [20], is a group1 protein and would be locted near the promoter of such a large ribosomal protein operon. Proteins S5 and 512 which correspond to the s p A and strA loci [20] are group I11 proteins and would be located between the middle and the promoter distal end of a ribosomal protein operon. According to our order of synthesis, S5 and S12 are closely related. This is substantiated by genetic studies [21] which have shown that the spcA and strA are cotransduced at a high frequency. The fus loci determines the elongation factor G protein. Our previous studies (unpublished) showed that the elongation factors are synthesized a t a lower rate than ribosomal protein during a shift-up. In this study, l4CI3H ratios of 0.074, 0.075, 0.078 and 0.080 were obtained for the elongation factors a t 0, 2, 4 and 6 min after the shift-up. These ratios indicate that the elongation factors were synthesized a t a rate lower than any ribosomal proteins (Tables 4 and 5) a t these times. These data also fit the order of synthesis suggested by Nomura and Engbaek [la] in which the cistron for elongation factor G would be located a t the promoter distal end of a ribosomal protein operon.

Although i t is possible to construct other models of ribosomal protein cistrons involving more than one operon [22], the experimental evidence for such models is not sufficient to warrant such an exercise a t this time.

Ribosomal Protein Synthesis and Assembly Gupta and Singh [23] and Beatty and Wong [24]

have suggested that the synthesis and assembly into subunits of ribosomal proteins are coordinated events. The groups of ribosomal proteins obtained as discus- sed above were analyzed to determine whether the proteins synthesized at the highest rate during a shift-up can be correlated with the proteins which are assembled fist during subunit biogenesis. The order of synthesis of the 30-S proteins is compared to the assembly order of 30-S proteins in vivo as determined by Marvaldi et a$. [4] in Table 6. The results in this table show that strong correlations differing by no more than one protein between the order of assembly and rate of synthesis exists for proteins 520, 56, 816, 515, 88, S9, 513, S3 and S2. Less certain correlations which d8e r by two to four

Table 6. Correlations of the synthesis and assembly of 30-5 ribosomal proteins The order of proteins in the first column was derived from the data presented in Table 4. The order of proteins in the second column was taken from the data of Pichon et al. [4]. The solid lines indicate a strong correlation between synthesis and assembly. Dashed lines indicate a weaker correlation between synthesis and assembly

Rate of synthesis Order of (highest to lowest) assembly

(early to late)

s20 s20 s10 .--S18

S6 _/-- - - 814 _- - /_--- S16 S6

S16 817 S18-”- 515 515 58 58 s4 s 9 s10 s7---- s 9

_ _ _ _ -- -- -s5 xi9 s19

-S7 512 __-_---- - ----I----- 5 5 - 5 - Sll------ 512

- ------s11 54 --- - -__

-- --- -- -__ - ---

Sl-, ------ - - _ _ _ _ _ _ 514 513

813 -_ -- 53 - - s 2 - -sl

-- -. ---_ 517 s 3 52

--- 521

proteins in their order of assembly and rate of syn- thesis are found for proteins S18, S7, S5, S l l and S1. No correlations were found for proteins S10, S14, S2 and S7. This analysis suggests that there is a coordina- tion of 30-S protein synthesis and the assembly of these proteins. Correlation between the rate of syn- thesis and order of assembly of 50-8 proteins was less obvious. The assembly data of Pichon et al. [5] did not include eight 50-S proteins which they were unable to isolate. The arrangement of proteins, based upon the synthesis data in Table 5, excluded nine 50-5 proteins which could not be reliably placed. Consequently, it was possible only to compare the rate of synthesis and order of assembly for 18 of the 34 proteins of the 50-5 subunit (Table 7). The results in this table indicate that strong correlations exist between synthesis and assembly for six 50-S proteins and less certain correlations for six other proteins. These limited results indicated that there is a weak correlation between the synthesis and assembly of the 50-5 proteins. Further investigations of the synthesis and assembly of the 50-5 proteins will be necessary before definite conclusions can be made about the coordination of synthesis and assembly. As discussed by Nomura [l], the assembly of the 50-5 subunit is more complex than that of the 30-5 subunit. Evidence has also been presented which

Eur. J. Biochem. 44 (1974)

130 G. Carpenter and B. H. Sells: Synthesis of Ribosomal Proteins during a Nutritional Shift-Up

Table 7. Cornelations of the synthesis and assembly of 50-8 ribosomal proteins The order of proteins in the first column was derived from the data presented in Table 5. The order of proteins in the second column was taken from the data of Pichon et al. [5]. The solid lines indicate a strong correlation between synthesis and assembly. Dashed lines indicate a weaker correlation between synthesis and assembly

Rate of synthesis Order of (highest to lowest) assembly

(early to latc)

L25 L25

indicates that the assembly of 50-S subunits may be dependent in some way upon the 30-5 subunit [l].

These studies were supported by the Damon Runyon Memorial Fund, the Medical Research Council of Canada and the National Foundation-March of Dimes. One of the authors, Graham Carpenter, was a predoctoral student at the University of Tennessee. The expert technical assistance of Miss Martha Hiegel is acknowledged.

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1276.

G. Carpenter and B. H. Sells, Laboratories of Molecular Biology, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada

Eur. J. Biochem. 44 (1974)