J. Mol. Biol. (1991) 222, 265-280

Absolute in lGvo Translation Rates of Individual in Escherichia coli

Codons

The Two Glutamic Acid Codons GAA and GAG Are Translated with a Threefold Difference in Rate Michael A. Smensen and Steen Pedersen Institute of Microbiology, University of Copenhagen Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark (Received 7 January

1991; accepted 15 July

1991)

We have determined the absolute translation rates for four individual codons in Escherichia coli. We used our previously described system for direct measurements of in viva translation rates using small, in-frame inserts in the EacZ gene. The inserts consisted of multiple synthetic 30 base-pair DNA oligomers with high densities of the four individual codons, GAA (Glu), GAG (Glu), CCG (Pro) and CGA (Arg). Our method is independent of expression level. of mRNA half-life and of transcription rate. Codon GAA was found to be translated with a rate of 21.6 codons/second whereas codon GAG was translated 3*4-fold slower (6.4 codons/s). These two codons are read by the same tRNA species. Codon CCG and CGA are both read by abundant tRNA species but nevertheless we found them to be translated slowly with rates of 58 and 4.2 codons/second, respectively. The context of these codons were varied, but we found no significant influence of context on their translation rates and we suggest a mechanism for why context may not affect translation rates. One insert with a low translation rate gave results that most readily can be explained by assuming queue formation of ribosomes on the insert. Such a queue was found to reduce the expression level by approximately 35%. Our experiments allowed us to est,imate the average distance between ribosomes and thereby the translation initiation frequency on the wild-type la& mRNA. This was found to be one per three seconds. Keywords: translation

rate: codon: context; ribosome; E. coli

weakly expressed proteins. The codon usage was recently shown to be the major, if not only, determinant of differences in translation rates (Sorensen

1. Introduction Knowing the translation rate of individual codons is necessary for understanding processes like translation initiation and global cellular regulation. In Escherichia coli synonymous codons are used with varying frequencies in different mRNAs. In particular, mRNAs for ribosomal proteins and other highly expressed proteins use a subset of codons almost, exclusively (Post et aZ., 1979; Grosjean & Fiers, 1982; Sharp & Li, 1987), the so called common codons. The concentrations of tRNAs in E. coli have been determined and a correlation has been found between the frequency of codon usage in highly expressed genes and the concentration of the cognate tRNAs (Ikemura, 1981a). A correlation between tRNA concentration and translation rate was found by Varenne et al. (1984). Pedersen (1984a) showed that mRNAs for highly expressed proteins were translated faster than mRNAs for 0022-2836/91/220265-16

$03.00/O

et al., 1989).

It has been suggested that the use of infrequent codons in the lowly expressed genes was selected to reduce the translation efficiency and thereby give a low expression level. This model has been rejected by Holm (1986) and Sharp & Li (1986) on the basis of frequencies of codon occurrences and the fact that it is the translation initiation frequency on each mRNA that determines the expression level (Holm, 1986; Sorensen et al., 1989; for a review, see Andersson & Kurland, 1990). The view is now that common codons are selected in highly expressed genes to increase the translational efficiency of these mRNAs (Kurland, 1987). By expressing tRNAs in proportion to this codon bias, a high translation rate on these mRNAs, particularly at fast growth, can be obtained for a relatively low investment in 265

0

1991 Academic

Press Limited

266

M. A.

Serensen

the translational apparatus per mass. Such an arrangement benefits organisms, like E. coli, that are selected for high growth rates (Ehrenberg & Kurland, 1984; Kurland, 1987). Common codons were proposed to have a high selection rate of their tRNAs due to high cognate tRNA concentrations and this should cause an increased translational efficiency (Ikemura, 1981b: Gouy & Gautier, 1982). It has also been suggested that an intermediate and optimal affinity between the codon and anti-codon gives this increased translational efficiency (Grosjean & Fiers, 1982). The correlation between codon usage and tRNA concentration is good, but not absolute. Codons read by the same tRNA are used with very different frequencies and even a rarely used codon may have an abundant cognat,e t,RNA (Ikemura, 1981a). There are many reasons why the translation rate might not solely be determined by the tRNA concentration. First, the chemical nature of the codons are different and even if tRNA modifications (for a review, see Bjiirk, 1987) presumably can do much t,o adjust the affinity between the codon and anticodon, affinity differences may persist, and this has been observed (Grosjean et al., 1978). Second, it is t,he sum of the concentrations of both the charged and uncharged form of the individual tRNAs that have been measured (lkemura, 1981a). We do not know the degree of amino-acylation for each individual tRNA species in vivo and a tRNA synthetase might charge each tRNA isoacceptor species with different efficiency. Third, it has been found that the affinities between elongation factor Tu and the different charged tRNAs are different. This seems to result in concentration differences of the ternary complexes that are not as la,rge as the differences in tRNA concentrations (Jakubowski, 1988). Finally. the amount, of uncharged tRNA may influence the translocation process (Brown, 1989; Rojiani et al., 1990) through an interference with t’he ribosomr E-site (Robertson & Wintermeyer. 1987). Recently, attempts have been made to measure codon-specific rates either in vitro (Thomas et al.. 1988) or in viva by the use of systems where differences in codon-specific translation rates are coupled to differences in expression yield (Bonekamp et al., 1989: Curran & Yarus, 1989). However, in vitro systems lack the charged and uncharged tRNA species that may be the natural in 21ivo competitors for binding to the A or E site on the ribosome and the coupled systems do not give the absolute codonspecific translation rates. The time it takes a ribosome to clear the ribosome binding site will affect the binding of the following ribosome and may be affected by codon usage (Sorensen et aZ., 1990). The absolute translation rates must, therefore be known to estimate the influence of codon usage on initiation rates. In addition, the overall chain growth rates of RNA and of protein polymerization may play a major role for the growth rate regulation in E. coli (Jensen & Pedersen . 1990). Previously, we developed an in viva method for

and S. Pedersen the accurate determination of the translation rate of’ inserts late in the la& gene (Sorensen et al.. 1989). We were able to show that t)he t,ranslation time of 24 codons, frequently used in highly expressed genes, is more than twofold shorter than that of the same to give rise to a short,er sequence. frame-shifted insert, containing infrequently used codons. From these measurements we estimated that at least, a sixfold difference in translation rate existIs among individual codons. but we were not able to give t,he exact) translation rate for any individual codon. Here we use this system to measure the translation rate on sequences with high densities of four specific, codons, inserted lat,e in the la& gene. We report’ a determination of the codon-specific translation rat,es for the two glut#amic acid codons GAA and (:A(: (exemplifying 2 codons read by t>he same tRNA) for the arginine codon CGA (representing an infrequent codon read by an abundant, tRNA species) and of the common proline (hodon CCC (to be able t,o compare the rates of 2 common codons).

2. Materials and Methods (a) Ntraim The strain used is SP536 (H0rensen ct al., 1989). derived from E’. coli K12 strain XAC (Miller et al., 1977). The genotype is A(lac-pro) rrcA uryG TnlO close to argG and

found (this work) to have the relaxed phenotype and to he resistant to nalidixic acid (most likely relA and CJ.~/T~. respectively). The strain carries the episomr F’ 1~~1~’ la& :: Tnj proAB+ from which the lac repressor is overproduced at a level t,hat) can repress the Zac operat’or cloned on multi-copy plasmids. SP536 is phenot~ypically Lacydue to the polar effect of the Tn:i insertion. This strain. SP536, harbouring different pBR322-derived (Bolivar et al.. 1977) plasmids wa,s used in the translation rate measurements. (b)

Plasmid

constructions

All in vitro DNA manipulations were’ in essen(‘e prrformed as described by Maniatis et al. (1982). I)fi;I restriction enzymes. ligase, polymerase (large Klrno\r fragment) and polynucleotide kinase were used as revonmended by the manufacturers. The construction of pMAS2. an amI)icillin-resistailt pBR322 derivative with the wild-type lac’Z2 Y’ sequences

(lack and lac Y partially

deleted) and its derivative

wit,11a

12-base-pair XaZT linker at the position equivalent) to codon 927 of la& (pMAS23) were both described previously (S0rensen et al., 1989). Synthetic oligonucleotides were a gift from Professor A. E. Dahlberg or purchased from commercial manufarturers. Electrophoresis of the oligonucleotidex was performed on sequencing gels to purify the maximal length of 30 bases. Approximately 10 pg of complementary strands were mixed after phosphorylat,ion by polynucleotidr

kinase and ligated

at room temperature.

Aft’er ligation

into fragment lengths with the average size of approximately 500 base-pairs, the DNA was extracted with phenol/chloroform and precipitated with ethanol. The DNA was then cut with 44.~~1 and HinPT to obtain concatemers of the linkers orientated only head to tail (see Fig. 1) and 90 or 180 base-pair long fragments were

Absolute Codon-specific Translation

Rates in E. coli

267

(a) Example linker

of 30 bp

Example ligated

of randomly linkers

‘??? ‘%?‘8 I: GCT (CTT,, GGC w CGCGA( GAA)8 &X$X ( TTC)8 TCLWGA ( GAA)8 CCGCGA(G?W8 C Mspl

(b) pMAS23

pMAS-24GAA

ATG TAT Ccc

3&F

c (GML)~ CCG CGA (GAA), CCC CGA (GTW8 CCC4ACC

GAT ACC

pMAS-4 8GAG

ATG TAT CGG

(cAo43cco =

c (-),

=G *

=

(-)8

y&P CGA (GAG)8 CCG CGA (CW)8 CCG CGA (GILG)~CCG ACC GAT ACC

pMAS-12 (GAACCG)

pMAS-12 (GAGCGA)

CC%

c ATG TAT CCC TCG CGA (GAA CCG)4 Ccc CGA (GAA CCC), CCC N&-U1 t CGA (GAA Ccc& CCG ACC GAT ACC

‘) ATG TAT CCC TC6 CGA (GAG CGA)4 CCG CGA (GAG CGA)4 CCC NrUI_ c CGA (GAG CGA)‘, CCG ACC CAT ACC

Figure 1. In vitro synthesized DNA sequences and the final inserts in the ZucZ. (a) Example of 30 base-pair linker (both strands shown) and the 3 possible internal orientations in a multimer of ligated linkers (1 strand shown). Arrows indicate the orientation of 30 base-pair linkers. Cleavage sites for the restriction enzymes MspI and H&PI are indicated. Digestion with these enzymes leaves linkers orientated “head to tail” with the original 5’ CG overhangs. (b) Sequences of typical inserts in the wild type la& gene of pMAS2. Inserts are shown by bold face letters. The non-template DNA strand is shown and the reading frame in the mRNA transcript is indicated by spaces. The pMAS23 opened with AccI was used for construction of the plasmids shown below with inserts of orientated linker concatemers (indicated by arrows). The NruI restriction site used for screening for orientation of inserts is indicated. The sequences of pMAS24GAG and pMAS-12(GAGCCG) are analogous to those shown for pMAS-24GAA and pMAS-12(GAACCG), respectively, just with GAG replacing GAA. The sequence of pMAS-48GAA is analogous to that of pMAS-48GAG. just with GAA replacing GAG. purified from agarose gels. These fragments were inserted in the Sail-AccI linker of pMAS23, first cut with AccI to yield compatible 5’ CG protruding ends. Sequences of the Inserts are shown in Fig. 1 or described in the Figure legend. All inserts were sequenced either by the method of Maxam & Gilbert (1980) or with modified T7 DNA polymerase (Tabor & Richardson, 1987) in the dideoxy-chaintermination method of Sanger et al. (1977). (c) Growth

and ZabeZZing of cells

The cells were grown in the minimal medium A+ B (Clark $ Ma&e, 1967) supplemented with @4% (v/v) glycerol, 50 pg arginine/ml, 2.5 pg thiamine/ml, 20 pg kanamycin/ml and 100 pg ampicillin/ml at 37°C. The

doubling time in this medium was 85 min. In the experiments where the medium was supplemented with 50 pg glutamic acid/ml the doubling time was 80 min. At A,,, between @4 and @5 (measured in an Eppendorf photometer model 1101M) 1@5 ml of culture was induced with 1.0 mM or, where indicated, @03 mw-isopropylthiogalactoside (IPTGt). After 2 min @2 mCi of [3H]lysine (88 Ci/mol) was added. The radioactivity was calculated to be completely incorporated after a few minutes, but to prevent residual incorporation a loo-fold excess of unlabelled lysine was added 14 min after the induction. One minute later, i.e. 15 min after induction, the cells were labelled with carrier-free [35S]methionine t Abbreviation

used: IPTG, isopropyl-thiogalactoside.

268

M.

A. kkwensen

(0.02 to 005 mCi: 1000 Ci/mmol). This pulse was terminated 10 s later by a chase with 0.1 mg unlabelled methionine/ml, shown previously to stop further incorporation within I or 2 s (Pedersen. 19846). Samples (0% ml) were taken at 6 to 10 s intervals into ice-cold t)est tubes containing chloramphenicol to a final concentration of 2 mg/ml. The cells were then spun down at 10,000 g for I min and the pellets frozen at -20°C’. Total protein extracts were prepared by boiling with SDS buffer. /?-Galactosidase was separated from all other proteins by electrophoresis on 7.5 9;, (w/v) polyacrylamidr stacking SDS-containing gels. After autoradiography. the p-galactosidase protein was cut out and the radioartivity measured (Pedersen el al.. 1976). An autoradiogram, made after cutting of the /?-galactosidase bands. is shown in Figure 2. In at least 1 experiment of each of t,he fi-galastosidase variants with altered mobility, the separations were also done by 2-dimensional gel elrctrophoresis (described by Pedersen ct al., 1976). This was done to ensure that results were not. influenced significantly by a position-specific background in the 1-dimensional gels. The method for determination of insert-specific translat,ion times was described previously (Scnrensen uf 4..

1989) and is summarized in Results. The theoretical

curve

best describing a set of data points was found among theoretical curves (see Results) calculated with a distance of 0.1 s. When repeated det,rrminations of the translation time of an insert) gave ident)ic-al results. t’he standard err01 was set to @I s.

((1) @uan,titative assessmrnf

oj radioactivity

by .scanniny

Autoradiograms to be scanned were made using Structurix X-ray film (Agfa-Gaevert). This film was shown to give proportionality between radioactivity and intensit,v on the film in the range used (J. Skouv, thesis. C.nivers&v of Copenhagen). Bands with large differences in intensities could be compared by scanning of autoradiograms exposed in different periods of time. The aut’oradio-

and N. Pedersen

grams were scanned using a Zeiss equipped with an integrating plotter.

514QIII

scanner

absolute

trans-

3. Results Our

method

for

determining

the

lation rate of individual codons is a refinement of the method described by Bremer & Yuan (196X) and Causing (1972). The translation time of small insert,s late in the 1acZ gene can be determined with a standard deviation below one second as described in detail previously (Sorensen et (IA.. 1989). Briefly. cells growing in the st!eady-state of induction are labelled with a pulse of [35S]methionirlr that is much shorter t,han the total translat,ion Gme. The pulse is terminat,ed by a cbhase. followed by rapid sampling. This allows a determination of the kinetics of the appearance of 3JS radioactivity in t,he finished protein product). The accumulation of radioactivit,y stops when the most N-terminal Label14 mrthionine residue has bec*n c>hased int.o full-size &galartjosidase. The t,ime-course of the, incorporation can he observed in Figures 2, 3 a,nd 4. We convert t’he amount of radioactivity at each point into the numbrr of codons translat,ed in the time between the pulse and the timca of sampling. The final amount of 35S in fi-galact, osidase corrf: sponds to the 23 methioninr residues in this protein, The number of methionine codons ira,nslated for each time point (*an t,hereforr br c*alculat,ed I,> dividing the sampled radioact,ivit8y by the final radioactivity and then multiplying by 23. Kec*ause we know the position of t.he methioninr codons from t)he 3’ end. the number of’ codons translated in each point caan be estimat,ed hy knowing the number of’ codons translated at the timr of’ methioniml

a’ a R- gal* R- gal*

Figure 2. A section of an autoradiogram of a preparative SDS-containing gel from an experiment where strains were mixed. From left to right the samples were taken at IO s int,ervals containing pMAS23 and pMAS-12(GAGCCG) starting 20 s after addition of [35S]methionine. The bands of the RNA polymerase subunits p and 8’ are indicat,ed. The 2 forms of B-galactosidase encoded bv pMAS23 and pMAS-12(GAGCCG), are indicated as /?-gal* and p-gal**. respectively. The clear spots in these bands originate from the sampling of protein for 35S/3H det,ermination.

Absolute Codon-spec$c Translation sampling. In addition, we use a double labelling procedure to be able to normalize to the same recovery of the fl-galactosidase protein in the samples (S0rensen et al., 1989). The methionine codons in the ZacZ mRNA are not in the equally spaced and the 3sS radioactivity completed j?-galactosidase will not rise linearly with time. To analyze the data, several theoretical curves are therefore calculated, each with varying average translation rate for the whole ZacZ mRNA. The curve giving the best fit to the data in a leastsquares analysis is then estimated. For the translation of wild-type 1acZ mRNA a rate of 12.45 amino acids per second was determined (Ssrensen et al., 1989). This value has been confirmed in the present work (12.4( kO.2) codons/s) and the theoretical curve best describing this rate is shown in Figure 3 (broken line). The inserts in the 1acZ mRNA for which we want to determine the translation time, are inserted after codon 927, which is 97 codons from the termination 1022 after the most codon (codon number N-terminal methionine in the mature protein). Two of the methionine codons in the ZacZ mRNA are downstream from the insert and therefore ribosomes translating downstream from the insert during the pulse brings the radioactivity into full-length /I-galactosidase with the same rate as on the wildtype mRNA. Ribosomes translating upstream from the insert during the pulse have to traverse the insert before they reach the 3’ end, and will be delayed by the time it takes to translate the insert. Compared to the curve for the wild-type, all points 25

I

20

t

Oc’l 0

” 20

” 40

” 60 Tirns

” 80

” 100

’ 120

(8)

Figure 3. Theoretical curves fitted to the data points shown in Fig. 4(d). Open and filled circles: appearance of radioactivity in fi-galactosidase incorporated by cells containing pMAS23 and pMAS-48GAG, respectively. Broken line, the calculated appearance of methionine incorporated from the wild-type la& mRNA with a theoretical average translation rate of 1245 codons/s. Full line close to open circles: the theoretical wild-type curve parallel displaced 3.0 s (after methionine no. 2) thereby giving the best fit to the data points from pMAS23. Full line near filled circles, the wild-type theoretical curve displaced 12.9 s. This displacement equals the cakulated time for translation of the total insert in pMAS-48GAG

(seethe text).

269

Rates in E. coli

sampled after the appearance of two methionine equivalents should be parallel displaced by the time it takes to translate the insert. This point of displacement can be seen in Figure 3. From this concept, we find the translation time of the inserts (Table 1) by finding the displacement from the wildtype curve, which gives the smallest sum of squared vertical distances between the curve and the actual points. The ZacZ gene is well suited for this kind of analysis because of its length, which allows us to sample many points before reaching the plateau value. (a) Translation rates of two synonymous codons read by the same tRNA We chose to insert repeated glutamic acid codons. either the GAA or the GAG codon, because these are the only codons for glutamic acid and they are translated by the same tRNAs (Ikemura, 1981a; probably only by tRNAy’” as the tRNA7’” does not seem to exist, at least in the E. coli K12 W3110 strain (Komine et al., 1990)). Furthermore, highly expressed genes have a preference for the use of codon GAA (Sharp & Li, 1986). To obtain EacZ alleles with the desired configura30 base-pair DNA tion , we inserted synthetic linkers that give mRNAs with a high local density of the codons we wanted to examine (Fig. 1). To give a sufficient translation time increase, we chose to insert the sequences three or six times resulting in inserts of 30 or 60 codons. As shown in Figure 1, the linkers are designed so that it is possible to make inserts of oriented concatemers into a site with 5’ CG protruding ends and to determine the orientation of the inserts by digestion with the restriction enzyme NruT. These demands make const’raints on the insert sequences: first, the proline codon CCC. and the arginine codon CGA are each present at least once per ten inserted codons and second, the naturally existing AccI restriction site with AT protruding ends, previously used for insertions (Scrrensen et al., 1989), had to be modified. This was done in pMAS23 by the insertion of a 12 base-pair linker into pMAS2, giving an AccI site with potential 5’ CG protruding ends and a 1acZ transcript with a supplement of the four codons CGG (Arg) UCG (Ser), ACC (Thr) and GAU (Asp). These four codons are conserved after insertion of the synthetic DNA concatemers (Fig. 1). To determine the translation rates of the sequences inserted into pMAS23, we measured the translation time of the insert in this parental construct. As stated below, pMAS23 was the reference plasmid in most of the experiments in which cultures were mixed. Numerous measurements (19) of the translation rate of the insert in pMAS23 show that the translation time is 2.9 seconds for the four codons. Examples of such experiments are shown in Figures 3 and 4. Figure 3 also shows the best theoretical curve describing one set of data. This time of 2.9 seconds can then be subtracted

translation

from the

time for the other inserts, giving

the

270

M. A. S’wrensen and S. Pedersen

translation time, AT, for the inserted codons. It is not unlikely that the rare arginine codon CGG and the infrequent serine codon UCG are each translated in approximately one second, and therefore account for most of the 2.9 seconds. The insert in pMAS23 is a palindrome and t’he corresponding mRNA has the potential t’o form a local secondary structure with the calculated free energy of formation, AG (calculated according to Freier et al.. 1986) of approximately - 10 kcal/mol (1 cal = 4.184 J). Our previous work (Sorensen et al. 1 1989) showed that much larger hairpin structures (AG of approx. -75 kcal/mol), in the same position of the la& mRNA, did not affect the translation rate and we therefore assume that t’his possible structure in pMAS23 does not delay translation. In preliminary experiments, we observed that the insertion of relatively few glutamic acid or proline residues into the /I-galactosidase considerably changed the apparent molecular weight of the protein in a one-dimensional polyacrylamide/SDS gel (Fig. 5). This enabled us to mix cultures before an experiment and subsequentl? after the experiment gel-purify the two b-galactosldase proteins (Fig. 2). The advantage of this is that differences in experimental conditions and sampling time between cultures to be compared, is now eliminated. The resulting displacement of t,he curves therefore dire&ly demonstrates the difference in translation time between two inserts. The pMAS23-containing strain was normally used as internal standard in the mixing experiments, e.g. in the experiments shown in Figure 4(a) to (e). Figure 4(f) shows the five pMAS23 curves in the same plot in order to indicate the reproducibility of the experiments. The experiments presented in Figure 4(a) and (b) show that there is a difference in the translation times of the inserts in the la& transcripts from pMAS-24GAA and pMAS-24GAG. These constructs have total inserts of 30 codons relative to the pMAS23 Zac.2 mRNA, but differ among themselves only in the use of either 24 GAA or 24 GAG codons (Fig. 1). The GAA insert is translated with a higher rat,e than the synonymous insert with GAG codons. To increase this time difference, the insert length was doubled in the constructs pMAS-48GAA and plMAS-48GAG and now, despite this doubling, the insert in the mRNA from pMAS-48GAA is translated in approximately the same time as the insert from pMAS-24GAG (Fig. 4). The results of all experiments are summarized in Table 1. Our determination of the insert translation time requires that, all ribosomes on average are delayed the same time by the insert, giving a parallel displacement of the curve. As can be seen in Figures 3 and 4(d), the rising part of the incorporation curve in pMAS-48GAG /I-galactosidase diverges more and more from that of pMAS23. We interpret this as being caused by a co-operative effect. A cluster of relatively slow codons increases the probability for a ribosome to be delayed so much as to cause a significant slowing of the following ribosome(s) (discussed below). In this case, all ribosomes are not

Translation

Table 1 times for the total numbers of inserted codons in our plasmids

Plasmid

Time for translation of insert, (s)

AT (s) -

pMAS23 pMAS-24GAA pMAS-48GAA pMAS-24C:AG pMAS-4&X4(: pMAS-lZ(GAOUX) pWAS-lI(QAGCC(:) pMAH-I”(GAAU’(:) Tanslation times for the total number of irwrted codons in OUI various plasmids (the codons indicated by bold face in Fig. 1). The numbers in parentheses show t)he number of experiments for each insert. The average translation time is given + the standard error of mean. The AZ’ value is the calrulated translation time fin the extra insert, in pMAS23 (tot,al translation time minus the translation time for thr 4 cotions originating from pMAS23) and its standard error of mean is calculated as the syuarr-root of t,hr sum of the 2 squared standard error of means. The values fol pMAS-48GAG are in brackets to indicate that t,his value ~~annol he used to estimate t,he translation rate due to the “queueinp effect” (see the text).

delayed the same time by the insert and we cannot use these results for a calculation of translation rate. Nevert,heless, the pMAS-48GAG result still demonstrates a qualitative difference in t’ranslation rate between GAA and GAG codons. (k)) Translation times fvr inserts u?ith rithw thr (‘PO or the (‘GA codon, alternating with ylu,tank ncid codons As discussed previouslv. the inserted sequences always have at, least one C’CG (proline) and one C($A (arginine) codon per t,en inserted codons. To csalculate the translation rate of any codon we therefore also have to measure the rates for t,he (I(‘(~ codon and the (“GA codon. To do so we inserted t’wo sequences: one with (‘GA alternating with (:A(: pMAS-12(GACCGA), and one having alternating CCC; and GAG (hodons pnirAS-l2(C:AG(:C(;). see Figure 1. The insert in pMAS-12(GAGCGA) t)hus contains 15 CGA, 12 GAG and three (‘CG cbodons and has t,he translation time (AT) of 60 seconds (Table 1). The insert in pMAS-12(GAG~‘CG) contains three (‘(:A, 12 GAG and 15 CCG codons and has a translation time increase of 5.2 seconds compared to pMAS23. To investigate if the translation times were influenced by the context of each codon, we also made pMAS-lB(GAACCG) where the insert cont’ains three CGA, 12 GAA and 15 COG codons. This insert. was found to be translated in 3.3 seconds (Table 1). (c) C’alculatiny six

the codon-specilc

translatior~

We have thus measured the translat’ion ins&s. having various combinations

rates time for of four

Absolute Codon-specijic Translation

271

Rates in E. coli

20

15

IO

5

a

pMAS23

0

pMAS - 24GAA

E

pMAS23

0

pMAS -48GAA

0

20

r .c .-6 f I”

15

IO

5

0

I

I

l

’

I

I

I

I

l

1

1

1

I

I

I

I

1

1

1

1

I

I

I

,

1

1

1

1

I

I

I

1

l

1

l

1

20

I5

IO

5

J

0

40

80

120

160

40

80

120

200

Time (s)

Figure 4. Appearance of radioactivity in j?-galactosidase incorporated by 5 different mixtures of cultures containing the plasmids indicated in each panel ((a) to (e)). The data points are connected with straight lines to facilitate an evaluation of the time differences in appearance of incorporated [3sS]methionine. (f) A plot of all the curves for pMAS23 from the 5 different experiments in the other panels.

codons, and we have used these to determine the translation time for each codon. As a first approximation we excluded the possible effect of context on the translation rate. It was then possible to calculate the translation time for the individual codons. To do this we used the four sets of data with the largest values of AT, where the experimental errors are relatively the smallest. These have also, by chance, the smallest absolute standard error of mean. If the translation time for GAA, GAG, CCG and CGA codons are TGAA, TGAG, TCCG and TCCA,

respectively, we then have equations from Table 1:

the

48T,,, + 6T,,, + 6T,,, 24TG,, i- 3TccG + 3T,,, lZTo,o + 3T,,, + 15TCGA 12TGAG+ 15T,,, + 3T,,,

following

= 4.7 s = 54 s = 6.0 s

= 5.2 s

four

(1) (2) (3) (4)

The solutions to these are given in Table 2. Using these values to calculate theoretical AT values for the remaining two inserts, pMAS-24GAA and pMAS- 12(GAACCG), gave 2.4 and 3.9 seconds

272

M. A. Scrrensen

Table 2 Translation

(:odon

times Translation (s)

GAA GAG (I(‘(” 1 Ir (“(‘A

and translation time

rates for codons Translation rate (codonsis)

0046 0.157 0.173 0.240

21.6 64 58 4.2

(lx-3-26.3) (5.9-69) (5-M) (3+-4(i)

Thr best estimate of the translation time and translation rate for the 4 codons GAA, GAG, CGA and C(G. calculated from the equations (l), (2). (3) and (4) in the text. In parentheses a.re indicated the span in the codon-specific translation rates, appearing when all analogous 4 equat,ions are solved using all combinations of the 4 AZ’ values + the standard error of mean (see the text).

respectively, only 0.6 second from the experimentally found values of 30 and 33 seconds (Table 1) and therefore within the limits set by the standard error of means. The fact, that the solutions to t,hese four equations gave consistent values, all in the range that is possible and reasonable for the cell, and that these values furthermore are in good agreement with t.he translation time for two other inserts, shows that, the translation rates of these specific inserts are not significantly influenced by context effects. The detailed argument that these translation rate differences are not caused by differences in the codon context is given in the Appendix. To examine the influence of the experimental uncertainty on the calculated translation rates, we generated the following 16 sets of equations analogous to the set above. The standard error of mean for each AT was calculated and either added to or subtracted from the AT values in Table 1. All possible combinations of AT f the standard errors results in I6 sets of equations, which were then solved. The resulting limits for the values of the four codon-specific translation rates are indicated in

I

2

3

4

5

6

and S. Pedersen

---.

Table %. Furthermore, the ratio between the translation rates on GAA and GAG is always between 2.9 and 4.0 and the t’ranslation rat’e of codon (:(:A is alwavs the lowest of the four rates. Only the difierence”in rate between (XX and (:A(: may not be significant. For all solutions to these 16 sets of equations. the calculated AT for thr insert in @IAS24GAA is between 2.3 and 2.4 seconds and the calculated AT for t,he insert of p~lAS-l2((:AA(!trc:) between 3.6 and 4.2 seconds. The experimentall> determined values 3*0( + 0.8) seconds and 3.3( + 0.6) seconds. respectively. are therefore in c*ompletr agreement with the codon-specific translation rat.es determined bv the other inserts. Because thr context differsin these inserts, we conclude that this has little, if any influence on the translation ratr. (d) Thr

Pzprcxsion leelel dors th4 tran.slation rate

not afert

The induction of a mR?JB with an unusually high content of a specific codon might generate an acute lack for a specific tRNA. This phenomenon has been report,ed in other systems especially when the codon in question was read by a rare tRNA (Misra 8 Reeves, 1985; Robinson d al.. 1984). Furthermore. such starvation might be enhanced if t,hr rare codons were sit,uated next to each other (Varenne &I Lazdunski. 1986; Varenne el al.. 1989; Spanjaard et al., 1990). To investigate if any art,ificial starvation is created in our experiments. we measured the translation rate in cultures where t)hr expression level was reduced by adding only 3 x 10 ’ YH-TPTG. This reduces the expression to %,5?,, of the full) induced level or approximately 2.5 times the fully induced level in a haploid strain (Sorensen of al.. 1989). The expression levels of such sub-induced cultures are shown in Figure 6. The translation time was measured during sub induction of cells containing the plasmids pMAS24GAA, pMAS-246AG. pMAS-12(GAWGA) and

7

0

(0)

9

I

3

2

4

(b)

Figure 5. Autoradiogram of 7.5% polyacrylamide/SDS gels on which total cell extracts from the used strains are separated (labelling conditions: 10 s pulse, 3 min chase). (a) In lane 1 no IPTG was added; in other lanes induction was with lWJ M-IPTG. The strains carried (lanes 1 and 2) pMAS23, (3) pMAS-24GAA, (4) pMAS-48GAA, (5) pMAS-24GAG. (6) pMA&48GAG, (7) pMAS-lB(GAGCGA), (8) pMAS-12(GAGCCG) and (9) pMAS-12(GAACCG). (b) Samples from experiments where 3 x IO-’ M-IPTG was used for the induction. The strains carried the plasmids: (1) pMAS-24GAA, (2) pMAS-24GAG,

(3) pMAS-lZ(GAGCGA)

and (4) pMAS-lL(GAGCCG).

Absolute Codon-speci$c Translation pMAS-lB(GAGCCG) and the results were the same as before, within the boundaries set by the standard deviations, and are included in Table 1. Also the shape of the diverging curve obtained with pMAS48GAG was independent of the induction level (J. Vind, unpublished results). From this we conclude that even full induction of these ZacZ derivatives has no detectable effect on the tRNA pools and does not disturb the general cell physiology during our experiments.

(e) Exogenous glutamic acid does not increase the translation

rate

We wanted to investigate to what extent supplementation of the medium with glutamic acid influenced the translation rate. Cultures containing either pMAS-24GAG, pMAS-48GAA, pMAS-48GAG or pMAS23 were grown exponentially with 50 pg glutamic acid/ml added to the medium. Experiments were made on cultures containing either pMAS-24GAG, pMAS-48GAA or pMAS-48GAG mixed with an equal number of pMAS23-containing cells. The translation time for the insert in pMAS23 was found to be 2*8( kO.3) seconds, 8.0 seconds for pMAS-24GAG, 7.5 seconds for pMAS-48GAA and the curve for pMAS-48GAG still diverges to the same extent as previously giving the value 15.8 seconds as the best value for the diverging curve. These values are not significantly different from the values given in Table 1, where no glutamic acid was added to the medium. We conclude that exogenous glutamic acid has no significant effect on the charging level of the tRNAG’“.

(f) Polysomes

with

queues of ribosomes

As mentioned above, we found that the insert in pMAS-48GAG gave a diverging curve for the appearance of radioactivity in the finished protein product (see Figs 3 and 4(d)). We interpret this as a co-operative effect of a cluster of slowly translated codons in the mRNA. The translation rate on the insert is so low and the insert is so long that there is an enhanced probability of formation of a ribosome queue during the translation of an average 1acZ mRNA. Before we discuss curve shapes and ribosome queues, we want to emphasize that the period spent by the ribosome on each codon, the time between two translation initiations on an mRNA, and the life span of an mRNA, all have a stochastic element in them due to their dependence on diffusion. Therefore, the values we normally refer to as translation time, translation initiation frequency and mRNA half-lift?, are averages of events with a certain degree of variation. The probability of two ribosomes encountering each other on a polysome is therefore dependent on the deviation for each of these processes. There will always be a probability for queueing of ribosomes on an mRNA. In the case of the insert at the 1acZ mRNA, if the translation

Rates in E. coli

273

rate at the insert is faster than, or equal to: that of the surrounding EacZ mRNA (12.45 codons/s) it will not change the pattern of queueing in the rest of the polysome and the shape of the curve should not be different from the wild-type. If, on the other hand, the translation rate at the insert is lower than at the upstream sequences, there will be an enhanced probability of formation of a queue at this particular site on the mRNA. This probability depends not only on the translation rate but also on the length of insert (related to ribosome diameter), the distance between ribosomes (initiation frequency) and the age of the polysome (this is because the first ribosome translating the insert below the average rate may initiate queue formation). In our experiments we have inserted codons close to the 3’ end of the 1acZ mRNA. We will first discuss the case where an insert has a high probability of queue formation during the life span of a normal polysome. If we look at the translation of the average 1acZ mRNA pool during such an experiment, the ribosomes incorporating [35S]methionine most 5’ on the mRNAs during the pulse will have the highest probability of encountering the longest queue of ribosomes in the 3’ end of the mRNAs. This means that the last ribosomes to finish translation, on average, have been delayed the most by the insert and this will precisely produce the curve shape seen in Figure 4(d) for pMAS-48GAG. For none of the other inserts we have used here, have we seen this phenomenon, which we interpret as caused by queueing. It is puzzling why such a queue is not’ generated by the pMAS-24GAG insert. This insert is 34 codons (compared to the wild-type sequence) and should therefore be equivalent to approximately three ribosome diameters (each 10 to 12 codons; Gold et al., 1981; Roland et al., 1988). The insert in pMAS-48GAG is 64 codons, equivalent to approximately six ribosome diameters and has almost the same codon composition. The average translation rate should be identical for bot’h inserts and the average distance between translating ribosomes should be the same. However, by doubling the length of the insert, the probability for a single ribosome to translate one or more codons with a rate much below the average is also doubled and this increased probability seems to be enough to create a ribosome queue on the insert in a detect’able number of the Zac-polysomes (diverging curves). Once a queue is formed, it will grow rapidly, because the queue will prevent fast translation of codons and thereby lower the average rate even of fast codons upstream from the insert. Although we have no direct evidence for the occurrence of ribosome queueing on the pMAS48GAG lac mRNA, we think this interpretation gives the only plausible mechanism behind this result. Furthermore, other unrelated sequences with the expected total translation time of approximately ten seconds give diverging curves (unpublished observations). That ribosomes can form queues on natural mRNAs has been demonstrated in vitro (Wolin & Walter, 1988) and also in wivo in

274

M. A. &wensen

E. coli during amino acid starvation (Dahlberg et al., 1973). The expression level of the various 1acZ alleles were quantitatively determined by densitometric scanning of autoradiograms similar to that shown in Figure 5(a). The amount of hybrid a-galactosidase protein was normalized to the amount of RNA polymerase subunits, fl and /I’ in each lane as previously described (Srarensen et al.: 1989). The expression level of b-galactosidase from pMAS48GAG is reduced to 65( + 10) y0 of the expression from the other alleles, which all are similar to the 13% of total protein produced by the wild-type 1acZ gene carried on pMAS2 (Smrensen et al.. 1989). Under the assumption that the mRNA half-life is not affected by the insert’, the approximately 357, reduction in expression level must be caused bv events like reading frame-shifts or premature termination. On the other hand. because we observe diverging curves, a significant fraction of t,he ribosomes encountering a queue makes full-length protein. If the reduction in expression level and the phenomenon of diverging curves observed with pMAS-48GAG are caused by the same mechanism, interpreted here as queue formation of ribosomes, this may be one of the mechanisms behind “processivity errors” e.g. reading frame-shifts or premature translational terminations in natural sequences found by Manley (1978) and Jorgensen CCKurland (1990), because such queues for stochastic reasons also may form on natural mRNAs.

(g) Determination frequency

of the translation initiation on the la& mRNA

The average initiation frequency on t’he 1acZ mRNA determines the average distance between the ribosomes. The average distance can be estimated by knowing the ribosome diameter (10 to 12 codons) and by comparing the translation rates on the insert that has the lowest translation rate without giving a detectable queue formation with the insert that does give a queue. The most slowly translated insert that does not give a diverging curve is the insert in pMAS-lS(GAGCGA) (see Fig. 4(e)). The total insert’ in pMAS-lB(GAGCGA) is 34 codons and is translated in 8-9 seconds giving t’he average rate of approximately four codons per second. If we assume that the ribosomes here are close to the limit for queue formation, the average time for translating a distance equal to a ribosome diameter should equal the time it takes the following ribosome to catch up. Assuming a ribosome diameter to be 12 codons, the average time between ribosomes will therefore be three seconds. The lower limit for the initiation frequency can be obtained from the translation rate on the insert in pMAS-48GAG. This total insert of 64 codons can be calculated to be translated in 12.9 seconds from the values given in Table 1. A shorter theoretical insert of 34 codons having this long translation time will therefore certainly give a queue. Using the t,ime

and S. Pedersen limits of 12.9 and X.9 seconds for the t,ranslation ot 34 codons (with and without queue formation, respectively) and the values of 10 to 12 codons for a ribosome diameter. our results therefore show that the average time distance between ribosomes on the 1acZ mRNA is between 2.6 and 3% seconds. This initiation frequency est,imate is in very close agrerment with the value of one per three seconds found for the 1acZ mRNA by an independent method by Kennel1 B Riezman (1977).

4. Discussion (a) Direct

~measurements of in viva

translatior[

rates

This work is the first attempt to determine the absolute translation rate of individual codons in living cells. Our method directly measures the translation time for an inserted sequence in the 1acZ mRNA in steady-state of induction and is independent of the expression level down t)o the limit set by the signal/background rat,io when determining the incorporated radioactivity. The method measures the time it takes for the radioactivity incorporated at, the most upstream methionine codon during the pulse to appear in t’he finished protein and is therefore also independent of any variance in the t’imr period before the initiation. Any reduction in expression level due t,o sequence-provoked frameshifts and premature t’ermination is also without influence, as only the time for successful translations giving rise to full length protein is measured. This renders the method independent of introduced variances in mRNA half-life and polarity. Furthermore, we also consider the method independent of the transcription rate. Tf any insert.ed sequence increases the transcription rate it might increase the polarity. due to de-coupling. bul not affect the translation rate on successfully transcribed mRXAs. On the other hand, an insert, with a hypothetical site for RNA polymerase pausing will only decrease t,he average translat,ion rate significantly if t)he mRNA has a very short half-life and is translated by only very few ribosomes. All the 1aeZ alleles used here for rate determinations have the same expression level (Fig. 5) as the wild-type and therefore we expect their mRNA half-lives to br unaltered. All derivatives of the bat mRlr’A used here are therefore, on average, subject to approsimately 30 translations (Kennel1 & Riezman, 1977). Only the ribosome following intermediately behind the polymerase can be affected by polymerasr pausing. Delaying this ribosome at’ most adds a negligible time increment to the average of all t’ranslations unless t,he polymerase pause is longer than the time between two translation initiations. which is, on average, approximately three seconds (Kennel1 & Riezman, 1977). A transcription pause must be longer than six seconds if the contribution to the measured translation time should exceed 77;, (2/30) of all translations. We consider an in vim transcription pause of six seconds or more to be unlikely. Nevertheless, such a pause will generate a

Absolute

Codon-specific

ribosome queue and be detectable by the diverging curve shape as discussed above. No insert that produces this shape of curve was used for translation rate determinations. For these reasons we believe that the translation rates found in this study are a reliable reflection of the in vivo translation rates. (b) Two codons read by the same tRNA diner in translation rate The two codons GAA and GAG are decoded by the same tRNA (Komine et al., 1990) and therefore the approximately 3.4-fold difference in translation rate cannot be caused by a difference in the cognate t)RNA concentration. According to the model of Gouy & Gautier (1982) the intrinsic entry rate, before recognition, of the cognate ternary complex to the A-site should be the same for the two codons. Several other mechanisms might cause this translation rate difference and none of them is mutually exclusive. First. the initial recognition between the cognate ternary complex and the ribosome A-site can be different at the two codons due to the difference in codon :: anti-codon base-pairing (Grosjean et al.. 1978) or possibly due to more pronounced competition from non-cognate ternary complexes (Kato. 1990) at the GAG codon. Second, in the proof-reading reaction (Hopfield, 1974; Ninio, 1974) the glutamyl-tRNA at GAG codons may be rejected preferentially. although a calculation shows that 6646 of all cognate entries at GAG codons should be discarded if this were the sole mechanism. Third: the time for mRNA/ribosome translocation may be codon-specific. Here we note that there is increasing evidence for rRNA :: mRNA interactions during elongation that could influence translocation times (e.g. Trifonov. 1987: Spanjaard & Van Duin, 1988; Dahlberg. 1989). Finally, tRNAs may not be completely modified and subclasses of tRNAF’” with different modification patterns may exist and preferentially decode one of the glutamic acid codons, in which case the translation rate of each actually be tRNA concentrationcodon may dependent. Based on the in vitro observations of Thomas et al. (1988) we believe that the initial recognition step. including proof-reading, is one of the most prominent determinants of the difference found in the translation rate of GAA and GAG codons. Thomas et al. (1988) found that both the tRNAPh’ (anti-codon GAA) and tRNAi’” (anti-codon GAG) each bind to their Watson-Crick base-paired codon faster than they bind to the wobble-base-paired coodon and that, after binding, the time for peptide bond formabion is the same for the four codons (Thomas et al.. 1988). Here we define Watson-Crick base-pairing as perfect base-pairing between the sequences of codons and anti-codons, primary without taking contributions from base modifications in tRNAs into consideration. In our case the anti-codon of tRNAy’” is 5’.CCC-3’. and the Watson-Crick base-pairing codon GAA and we

Translation

275

Rates in E. coli

believe that the difference in translation rate is partially caused by affinity differences between the tRNA and the two glutamic acid codons. may be an additional parameter There influencing the difference in rate between translation of GAA and GAG codons. Recently, Kato et al. (1990) demonstrated that the decoding of the A-site codon is dependent on the type of basepairing in the P-site of the ribosome. They did this by changing in vitro the anti-codon GmAA to AAA changing the hyperof the yeast tRNAPh” without modifications of the neighbouring bases. They were able to show that poly(U) programmed ribosomes bind the native tRNAPh” (GmAA) most efficiently if the P-site is occupied by the Watson-Crick type base-pairing anti-codon AAA, compared to the wobble type base-pairing GmAA. Both combinations were shown to perform transpeptidation with the same efficiency. These data seem to exclude the possibility that it is tRNA-tRNA interactions at the ribosome that provide the effect, because, except for the anti-codon bases, the tRNAs are identical in every respect. Instead the base stacking of the codon in the A-site may be affected by the type of base-pairing in the P-site, especially to the 5’ neighbour base. Therefore, the affinity between the A-site codon and the next cognate ternary complex will probably also be affected by the complex in the P-site. Many authors (e.g. Bossi, 1983; Carrier & Buckingham, 1984; Murgola et al., 1984: Gutman & Hatfield, 1989; Smith & Yarus, 1989) have suggested that tRNA interactions a,t the ribosome are crucial for translation efficiency. The results of Kato et al. (1990) indicate that a difference in tRNA interaction can be mediated through a difference in the mRNA conformation. In our case the Watson-Crick type base-pairing between codon GAA and tRNA:i” may analogously increase the translation rate of the next codon, or alternatively the wobble base-pairing to GAG may reduce the translation efficiency of the following codon. As discussed in the next section. this does not imply any effects of context on the overall translation time for a codon sequence: meaning that only the codon content in a sequence, and not the order of codons. determines t’he t,otal translation time of the sequence. In conclusion, we believe that the difference in translation rate of the two glutamic acid codons is caused mainly by two components: an affinity difference between tRNA7’” and the two codons, and an effect on the recognition time for the next cognate ternary complex depending on the type of glutamic acid codon occupying the ribosome P-site. An in vitro determination of the affinity difference made in the same way as for tRNAPhe and tRNAp” by Thomas et al. (1988) would clarify this matter. (c) Context eSfects and translation If the P-site complex A-site, independently provides an explanation

rates

modifies the affinity to the of the A-site codon, it for why codon context has

276

M. A. Sorensen

little or no effect on translation rate, while numerous reports show context to affect the translation error frequency (Precup et al., 1989) as well as the efficiency of nonsense suppression (Bossi; 1983: Buckingham et al., 1990) and missense suppression (Murgola et al., 1984). The main difference between their experiments and ours is that they measure events principally related to A-site selection, while we measure total codon transit t’imes. Our method cannot, dist,inguish whether the codon-specific effect on t,ranslation rate is caused 1,~ having the codon in the A-site or in the P-sit,e, because we measure the total time it takes for t,he codon to pass the ribosome. Therefore we cannot determine if this is a, direct effect on the rate of tRPL’A entry of having the codon in the A-site, or a more indirect effect’ of having it in t’he P-site. In contrast. if the codon :: anti-codon binding type in one site affects the binding to the codon in the neighbouring site, a variation of the binding type in one site may well be crucial if one looks at rates (probabilities) for competing events in t’he other site. For example. one could compare. during missense suppression, the probability of entry of the suppressor tRNA with the probability of entr.y. of the cognate tRNA. Tf the P-site type of paumg affects the entry rate of one or the other tRNA. it will caausea measurable effect of upstream context (Murgola et al.. 1984). I n other words, we suggest that context may have litt’le effect on translation rat,e when measuring the additive translation time contributions from each codon’s stay in the A and P-site, whereas all events depending on t’he probability for success in just one site will show large context variability. The idea that the pairing t’o the third base in t’he P-site is crucial to the select,ion rate of the next tRNA is supported by the work of Buckingham et al. (1990) where they examine the six serine codons as context to a CGA codon and find that the third (wobble) base is an important determinant of the efficiency of lJGA suppression. Tt seems from their data (Huckingham et al., 1990) that upstream wobble pairing (on the codons AGU and LTCLJ) provides the least efficient context, to the suppressor tRNA compared to the other four serine rodons. In contrast, downstream context effects seem much more dependent on the neighbour base (suppression favoured by an A base) as also found in other cases (Kossi 1983: Miller & Albertini, 1983; Stormo et al.. 1986). This downstream context effect in nonsense suppression (Hossi. 1983) might be highly influenced by unique features of the termination process, such as involvement of termination factors and a possible base-pairing interaction between the terminat’ion codon and 16 S ribosomal RNA as shown for the UGA codon (Murgola et al., 1988) and suggested for the other termination codons (Murgola et al.. 1990). Tn conclusion, we suggest t’hat the Watson-Crick type of base-pairing enhances the translat’ion rate of the following codon, or that the wobble base-pairing decreases it. This may happen irrespective of the identit)y of the A-site codon, and context may

and 8. Pedersen

therefonx rate.

have a minimal

(d) Translation

influence on translation

rates and rodon

usayr

One may attempt, to estimate the absolute translation rates of the 29 sense codons that begin with I’ or (I, by using our estimat,es of the translation times for the codons CCG and CGA. The relative rates of aminoacyl-tRKA select,ion at the A-site have been determined for these 29 codons by C!urra,n B Yarus (1989). Thev measured t,he c*ompetit#ion between tRNA sele&on and reading frame-shift in a system adapt,ed from the mechanism of regulation of expression of release factor 2 (Weiss et al.. 19X8). A fairly good correlation between the codon usage in highly expressed genes and the rrlativtl rat,r of aminoaty-tRNA A-site entry was found (Curran & Yarus. 1989). L~nfortunately, the two codons (1(X: and CGA are. in the work of Curran and TanIs. the two that deviat-e most from the general correlation between codon usage and tRNA entry rat,r. (‘odor1 (K:A is very rarely used in highly expressed genes. but thr aminoacyl-tRNA:‘g ent’ry rat,e at this cxodon was found to he among the highest ((lurran 8r Yarus. 1989). WC find the translation rate of t)his codon to be low, i .A. 4.2 codons per secsond or three times lower than the average translation rate of t,h(l Znc% mRNA. So in the case of codon (‘(:-4. caodon usage in highly expressed genes correlates bet)tclr with translation rat,e t’han wit,h cognate tRSA entr!, rate. (lodon (‘CX is the most c*ornrnonlg used prolinr codon in highly expressed genes. but was found tjo have one of the lowest t)KNA entry rates at t.he ribosomr txven when compared to the rarely usrd prolinr (sodons (‘(X’ and C(‘1’ (C’urran 8 Yarus. 1989). There is a good oorrelat.ion bet’ween choice>of’ synonvmous codons in highly exprrsscd genes and h’igh iranslation rat,ths (Ikemura. 1!)816: Ped~~rsr~n. 19X4a: \:arenne pf al.. 1984: Kurland. 19X7: Sc?rensen rt al.. 1989) and therefore we yenrrally expect t,he fastest of t)hr synonymous codons to 1)~ the preferred oodon in highly expressed genes. This correlat,ion is a#lso found for thca ylut~arnic acid c~dons. where (:AA constitutes approximately 7S”,, of thus glutamit acid caodons in highly expressed genes (Sharp & Li. 19%) and we find (iA4 tra.ns~ lat)ed 3.4 times fast’er than (:A(: (Table 2). It is t,herefore likely that, even if the proline c~don (‘(Xi is t,ranslated slowly, it may well be thrl fastest of t,hcfour proline (sodons. For these reasons. w(’ do not caalculate the absolute translation times for other codons from t,hts relative tRXA enl r!’ rat,rs of’ Curran & Yarus (1989) and our data. Supplementing t,he growth mediurn with glutamic acid did not affect the t,ranslation rat,cl of’ an> glutamic acid codons. Wr cannot det~~rmine if t)hr translation rate of the arginine codon ( ‘(:A would he different if the medium had not been supplemrnted with arginine. because t)he strain used is an argininr auxotroph. On ihe other hand. like tRNA~‘” the tRNATrg is expected t,o be present in high CO~(VIItrntions (see below,) and bot)h tRStls rra,d codons

Absolute Codon-specific Translation preferred in highly expressed genes. tRNAfrB reads the three codons CGU, CGC and CGA by virtue of the inosine wobble base (Ikemura, 1981a; Komine et al., 1990)). We therefore do not expect a large effect from the arginine supplement on the translation rate, but further studies are needed to elucidate this matter. Recently, growth rate-dependent regulation of tRNA expression has been reported (Emilsson & Kurland, 1990; Fournier & McKay quoted in Andersson & Kurland, 1990). We note that the measurements performed by Ikemura (1981a) on tRNA concentrations were done in rich medium whereas our experiments are done in minimal glycerol medium. Also, strain-specific differences in tRNA levels have been reported (Jakubowski & Goldman, 1984). However, such variations should he drastic if the four codons examined in this work are not translated by abundant tRNAs in our strain and medium. Therefore, our work most probably demonstrates that a high concentration of a cognate t,RNA does not necessarily imply a high translation rate and, as mentioned in the Introduction, the reasons can be many. However, recognition of the large difference in the translation rate between the glutamic acid codons is important in order to understand the reasons behind bias in choice of synonymous codons.

Appendix In this Appendix we will argue in detail that it is the nature of codons and not the codon context that most significantly determines the observed translation rate differences. Tn general, there are two main models to explain context) effects. One suggests that tRNA interactions at the ribosome affect translation in a variable way, depending on the tRNA species in the A and P-site (e.g. Bossi, 1983; Murgola et al., 1984; Smith & Yarus. 1989). The other model is an extended codon model, which suggests that the identity of neighbour bases affects the codon :: anticodon binding (e.g. Shpaer, 1986; Folley & Yarus, 1989; Gutman & Hatfield, 1989). These models build on observations of nonsense and missense suppression and statistics on frequencies of neighbouring bases. In addition it has also been suggested that ribosomal RN4 :: mRNA interactions during translation have produced constraints on the natural mRNA codon composition (Trifonov, 1987: Spanjaard bz van Duin. 1988). First, the difference observed here in the translation rate of repeats of either GAA or GAG codons cannot be caused by differences in tRNA-tRNA interactions. This is because it is the same sequence of the same tRNAs entering the A and the P-sites during translation of these two inserts. This leaves two possible mechanisms of context to influence the translation rate of these two inserts. One is an effect on the translating tRNAy” of having either A or G as the neighbouring 5’ base. In this case it should (‘ause a glutamic acid codon to be translated slowly

Rates in E. coli

277

when having G, as opposed to having A, as the upstream base. Another possible context effect is that ribosomes might bind more tightly to a repeated GAG sequence, because the GAG repeat has a high affinity to the anti-Shine-Dalgarno sequence on the 16 S RNA (Spanjaard & van Duin, 1988). This question is addressed by the results with pMAS-lS(GAGCGA). Compared with the translation rate of GAA codons, the insert in pMASlB(GAGCGA) is found to be translated with a low rate (AT = 6.0 s), although the GAG codon here is always preceded by an A and the GAGGAG motive is interrupted. We can exclude the possibility that the neighbouring base context around the CGA codon is particularly delaying in this case. because in all our constructs the CGA codons are followed by a glutamic acid codon (GAA or GAG) and are always preceded by the base G. Therefore, the codon CGA in the same surroundings is also present six times in the insert in pMAS48GAA. and this is rate found the insert with the fastest translation (Table 1). If the low translation rate of pMAS12(GAGCGA) had to be explained by context, the most plausible mechanism, as opposed to the GAGGAG case, would therefore be tRNA-tRNA interactions at the ribosome. The other insert, pMAS-lB(GAGCCG), is translated in 5.2 seconds. This relativelv low translation rate, compared to the rate on the”insert in pMAS48GAA, could be low due to t’wo different context mechanisms: (1) the base G occurring 5’ to all the glutamic acid codons (also the possible reason for the low translation rate of the repeat of GAG codons); (2) an interaction between tRNAG’” and the tRNAP’“. Therefore we made one additional insert, pMAS-lB(GAACCG), where t’he sequence has identical neighbouring tRNA interactions compared to the pMAS-12(GAGCCG) insert and also has G upstream from the glutamic acid codon. The translation time for the insert in pMAS-12(GAACCG) is only 3.3 seconds (codons in insert: 15CCG. 12GAA and SCGA). This is 1.9 seconds less than the insert in pMAS-lS(GAGCCG). We therefore find it unlikely that a 5’ G neighbour decreases the translation rate of glutamic acid codons, or that a possible interaction of tRNAG’” and tRNAP“’ on the ribosome should by itself decrease the translation rate. We are aware that codon CCG in these two constructs is in different contexts, having either G or A as the 5’ base and this may offer a theoretical explanation for the observed translation rate differences. However, comparing these hypothetical requirements with the in viva occurrences of contexts, it is found that the proline codon CCG, with high significance, has G as the preferred 5’ nucleotidein highly expressed genes (Shpaer, 1986) and this should not be expected if it decreases the translation rate. In the same set of data, it is demonstrated that the GAG codon is preferred downstream from A and upstream from C or A, whereas GAA is t’he preferred codon both upstream and downstream from G in highly expressed genes (Shpaer. 1986). This neither

878

M.

A. Ssrensen

contradicts, nor substantiates the suggestion that neighbouring bases are the basis for the translation rates found on glutamic acid codons. On the other hand, the fact that GAA is the preferred codon for glutamic acid in highly expressed genes (Sharp $ Li. 1986) supports the idea that this preference is due to a higher translation rate. Tf differences in codon context were t,he explanat,ion for the observed translation rate differences, an almost infinite series of ad hoc explanations is required, involving different mechanisms in each case. Therefore, from all these arguments and data we find it very unlikely that differences in context by coincidence should balance and be the reason for t.he otherwise clear differences found in translat.ion rates on the various inserts. We conclude that in the cases investigated here the effect of codon context is small and undetectable by our method. We thank M. Warrer for excellent. technical assistancr. A. E. Dahlherg for the generous gift, of oligonucleotides. R. H. Buckingham. C. C. Kurland. C’. Petersen and C’. Squires for critical comments on the manuscript and G. Q. Penapple for his encouragement. M.A.S. received a fellowship from NOVO A/S. The work was supported b> grants from the Danish Center of Microbiology.

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