THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, , Inc.

Vol. 264, No. 29. Issue of October 15, PP. 17243-17249,1989 Printed in U.S.A.

The Nucleotide Sequence and Characterization of Four Chloroplast tRNAs from the Alga Codium fragile*

(Received for publication, May 2, 1989)

Marilyn A. Francis, Eun Ran Suh, and Bernard S. Dudock From the Department of Biochemistry, State University of New York, Stony Brook, New York 11794

The nucleotide sequences of four chloroplast tRNAs (methionine elongator, lysine, glycine, and arginine) from the siphonaceous green alga Codium fragile have been determined. These tRNAs have an unusually high A-U content compared to other chloroplast tRNAs and show varied, but in general only limited, sequence homology to the corresponding tRNAs of other chlo- roplasts. The locations of the genes for these four tRNAs have been determined and they show no simi- larity to the location of the corresponding tRNA genes in other chloroplasts. The Codium chloroplast glycine tRNA has an unmodified uridine in the wobble position of the anticodon, a characteristic rarely found in tRNA but present in mito’chondrial tRNAs which read the genetic code by extended wobble.

Chloroplasts, like mitochondria, contain their own genomes and protein synthetic machinery which are distinctly different from their respective nuclear and cytoplasmic counterparts (1-5). In recent year:;, much work has been done at the molecular genetic level to elucidate the structure and organi- zation of the chloroplast genome and protein synthetic ap- paratus (6, 7). By and large, most of these studies have been done on chloroplasts from land plants, including maize, pea, and spinach, and especially from tobacco and the liverwort Marchantia po1ymorph.a. Indeed, the complete nucleotide se- quences for both the tobacco (155,844 base pairs) (8) and M. polymorpha (121,024 base pairs) (9) chloroplasts have been determined. Both encode a similar number of different genes, a total of 119 in the liverwort and 122 in tobacco. These consist of 30 (tobacco) or 32 (M. po lymorph) different tRNA genes, and 4 ribosomal RNA genes (4.5, 5, 16, and 23 S rRNAs), with the remainder being protein coding genes.

Codium fragile is a siphonaceous green alga (Chlorophyta) which grows as large firm clusters in many parts of the world. It was accidentally introduced, probably from Europe, to the east coast of the United States in 1957 where it now grows in abundance (10, 11). Codium is taxonomically quite distant from those plants whose chloroplasts have been well studied, particularly higher plants, and its chloroplast genome con- tains a variety of unusual features. These include its unusually high A-U content and its very small size, only 89 kb’ (12, 13)

* This study was supported in part by Grant GM-25254 from the National Institutes of Health and Grant PCM-8201925 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EM,BL Data Bank with accession number(s) 505040.

The abbreviation used is: kb, kilobase pair.

compared to an average chloroplast genome of 120-160 kb, the absence of a large repeat region (13) characteristically present in most chloroplast genomes, and the observation that the gene organization of the Codium chloroplast genome shows essentially no similarity to the “consensus” vascular plant order or that of any other green algae or bryophyte chloroplast genome (13). In order to better understand chlo- roplast structure-function relationships as well as the diver- sity present within chloroplasts, a more detailed study of the Codium chloroplast system has been undertaken. In this paper we report the nucleotide sequences and characteristics of four Codium chloroplast tRNAs: tRNAGIYUCC, tRNALY”U*UU, tRNAArgICG, and tRNAMetCAU.

MATERIALS AND METHODS’

RESULTS

Codium Chloroplast RNAP-The nucleotide sequence of Codium chloroplast tRNApt (Fig. 1) was determined primar- ily through the application of the partial formamide hydrolysis method. Using this technique, as illustrated in Fig. 2, residues 1-70 were determined. Modified residues were digested with nuclease PI and identified using the two-dimensional thin layer chromatography system of Silberklang et al. (20). The chromatographic characteristics of modified residue mZG’O are shown in Fig. 3a. The unknown residue at position 37, desig- nated X37, exhibits a mobility of RpAp = 1.05 in the ammo- nium sulfate system of Gupta and Randerath (16). In the two- dimensional system of Silberklang et al. (20) pX migrates close to pA and similar, but not identical to pm6A (Fig. 3b). RNA sequence gels show that this residue is not cleaved by RNases TI, U2, or BC, but is cleaved to a significant extent by RNase PhyM, which cuts at U + A residues. Furthermore, in all methionine elongator tRNAs except those of mitochon- dria, position 37 is occupied by an A or modified A residue (25). It is therefore likely that residue X37 is a modified A. A modified residue with the same properties as residue X 3 7 is also found in position 37 of Codium chloroplast tRNAA’g.

A series of RNA sequence gels (20, 12, and 8% polyacryl- amide) provided confirmation of nucleotides 1-70 and allowed the identification of residues 71-77. In order to provide con- firmation of residues 71-77, mobility shift analysis was per- formed on [3’-3’P]tRNAp.

Codium tRNA? can be aminoacylated in vitro under standard assay conditions with 13H]methionine (and not with [3H]isoleucine (24)) in the presence of a crude extract of Escherichia coli synthetase. In addition, the methionine moiety of this aminoacylated tRNA was not retained upon

Portions of this paper (including “Materials and Methods,” Table I, and Figs. 2 and 3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

17243

17244 Characterization of Four Algal Chloroplast tRNAs

tRNA 'ys C A

pG-C G-U G-C - 70 U-A

pi

U-A A-U C - G

'O u v! U

G U A A D 2i A-U A

C- G 30- G-C-40

CG-' A

JI-A

U t6A U*" u

60 I U u A

C F c A G G G

m7G 55 I

Arm C C U

pG-C C-G A-U- 7O G-C U-A A - U C-G

lo u V F $ F $ viG A G G G G

c/c u-A A f"'6

u-48

26 U - A

30-G-C-40 A - U

tRNAgtY JI U A

tRNA

I ' A

G

T J I

55 I

A, C

A C

pG-C C-G

A - U U - A

U - A I U

C - G -70

60

10 U - A

C-G C- G

30"G-c--40 JI- G

A A

J I c A

I 55

A W C C A

pG-C G-U

U - A 60 A-U

IO A-U U - A L A A

G-C "70

I5

I ' G A

q v c i A ' G G G G G c u F F F G

G A G U

D u\ 26 A-U

20' 30-G-C -40

c-48 T c A ' A ?n7G .+ I / '-A A

55

I 20'' U - A

G- C

tRNAorg C A

U X

FIG. 1. The nucleotide sequences of four Codium chloroplast tRNAs.

treatment with CuS04 in formylation assays (26). Codium chloroplast tRNA,M"t or an oligodeoxyribonucleo-

tide corresponding to the 5' end of the tRNA molecule (GCCTATTTAGCT) were used as probes to hybridize to Codium chloroplast DNA restriction digests. These probes hybridized to a 10.0-kb XhoI fragment, a 8.1-kb BclI fragment, a 12.0-kb ClaI fragment, and a 5.6-kb EcoRI fragment (Fig.

-~ CODIUM FRAGILE CHLOROPLAST (89 K b )

*

W o R I / FIG. 4. Restriction map of Codium chloroplast genome.

4), and to a 16-kb HindIII fragment and a 28-kb BamHI fragment.

Codium Chloroplast tRNALys-The nucleotide sequence of Codium chloroplast tRNALy" (Fig. 1) was obtained primarily through the application of the partial formamide hydrolysis method. Using this technique, residues 1-72 were determined. However, as is sometimes the case in tRNA sequencing by this method (17), a band compression was observed corre- sponding to residues 46-50, making the proper nucleotide assignments difficult in this region. To overcome this prob- lem, an oligodeoxyribonucleotide (GGGTTACTAACTCA- ATGG) corresponding to the 5' end of the lysine tRNA was synthesized. This probe was 32P labeled and used to screen a Codium chloroplast EcoRI recombinant DNA library cloned into the plasmid pBluescript. Suitable clones were isolated and the Codium chloroplast tRNALy" gene was sequenced using the dideoxynucleotide terminated primer extension re- action (27). This tRNALy* gene sequence, to be discussed in detail elsewhere3 was in complete agreement with the RNA sequence and allowed the assignment of residues 46-50.

Modified nucleotides were confirmed by thin layer chro- matography in the two-dimensional system as described (20). The unknown modified U at position 34 runs close to U in ammonium formate and has an RpUp = 1.2 in the ammonium sulfate system of Gupta and Randerath (16).

Codium chloroplast tRNALyE or the oligodeoxyribonucleo- tide corresponding to the 5' end (see above), hybridized to a 7.2-kb XhoI fragment, a 9.0-BclI fragment, a 4.8-kb Ckd fragment, and a 5.3-kb EcoRI fragment (Fig. 4), and to a 7.0- kb BamHI and a 2.2-kb HindIII fragment.

Codium chloroplast tRNALy" is aminoacylated by E. coli synthetase in an in vitro aminoacylation assay.

Codium Chloroplast tRNAGIy-The 75-nucleotide long se- quence of Codium chloroplast tRNAGly is shown in Fig. 1.

M. Gidh and B. S. Dudock, manuscript in preparation.

Characterization of Four Algal Chloroplast tRNAs 17245

Residues 3-73 were determined by the partial formamide hydrolysis method. The 5' terminal G was liberated by com- plete nuclease PI digestion of [5'-32P]tRNAGly and identified by two-dimensional chromatography (20). Additionally, [5'- "P]tRNA"'Y was used in a mobility shift analysis for the identification of residue Cp. Confirmation of the sequence was provided by 20 and 12% polyacrylamide RNA sequence gels using [3'-"P]tRNAG'y.

As a result of slight band compression involving residues 50-52 which occurred in both the partial formamide hydrol- ysis gels as well as the RNA sequence gels, evidence was obtained for only 3 of the 4 G residues actually present in positions 50-53. The fourth G was unambiguously shown to be present by mobility shift analysis of a 5'-3ZP-labeled frag- ment whose 5' terminus was U48.

The U residue at position 34 was identified as an unmodi- fied U on the basis of its chromatographic mobility in four different solvent systems. As shown in Fig. 5, this residue migrates as an unmodified U in both the ammonium formate and ammonium sulfate systems of Gupta and Randerath (16),

a b

U C

A

U

G

C

A

J l A A C C U I

q A A C C U I

38 34 38 34 I I

C

L

L, FIG. 5. Verification of the unmodified U in the wobble po-

sition of the anticodon of Codium chloroplast tRNAGIY. The residue at position 34, the wobble position of the anticodon, of Codium chloroplast tRNAC'' was identified as an unmodified U in the am- monium formate ( a ) and ammonium sulfate ( b ) solvent systems of Gupta and Randerath (16) as well as in the two-dimensional solvent system of Silberklang et al. (20) (c).

as well as the two-dimension system of Silberklang et al. (20). Codium chloroplast tRNAGly can be aminoacylated in vitro

with crude extracts of either wheat germ or E. coli synthetase. Codium chloroplast tRNACIy or an oligodeoxyribonucleotide

probe corresponding to the 5' end (GCAGTACTG- GTGTAATGG) hybridized to a 26.0-kb XhoI fragment, a 2.7- kb BclI fragment, a 6.6-kb CluI fragment, and a 1.1-kb EcoRI fragment (Fig. 4) as well as to a 3.6-kb Hind111 fragment.

The gene for the Codium chloroplast tRNAGIy was isolated and sequenced in the same manner as described above for the Codium chloroplast tRNALY" gene. This tRNACIy gene se- quence, to be discussed in detail elsewhere: was in complete agreement with the RNA sequence.

Codium Chloroplast tRNA ArR-The nucleotide sequence of Codium chloroplast tRNAAw (Fig. 1) was determined primar- ily by the partial formamide hydrolysis procedure, from which residues 6-71 were determined. These residues were con- firmed by RNA sequence gels and, in addition, residues 34- 39 were also confirmed by mobility shift analysis. Finally, residues 1-5 and 72-76 were determined from RNA sequence gels and mobility shifts.

The modified residue at position 37 of tRNAA9, designated X, is the same as residue X37 found in Codium chloroplast tRNAp, and has been discussed above.

Codium chloroplast tRNAArK can be aminoacylated in vitro with crude wheat germ or E. coli synthetase.

Codium chloroplast tRNAAw, or an oligodeoxyribonucleo- tide probe corresponding to the 5' end (GGGTATATAACT), hybridized to a 6.7-kb XhoI fragment, a 4.7-kb BclI fragment, a 1.3-kb ClaI fragment, and a 0.6-kb EcoRI fragment (Fig. 4).

DISCUSSION

The four Codium chloroplast tRNAs which have been se- quenced all have an unusually high A-U content (53-58%, Table I) which is, except for the chloroplast tRNA? of land plants, higher than the A-U content of the corresponding tRNAs of other chloroplasts or E. coli. The high A-U content of these tRNAs perhaps reflects the high A-U content (63%) of the Codium chloroplast genome itself (12). These Codium chloroplast tRNAs show quite varied, but in general only limited, sequence homology to the corresponding tRNAs of other chloroplasts (Table 11). At one end of the homology range is the Codium chloroplast tRNAGlyUCC, which shows only 65-66% homology to the other land plant chloroplast tRNAG'YUCC isoacceptors. This level of homology is quite low, considering the presence of 20 conserved or semi-con- served nucleotides (not including the CCA end) present in essentially all tRNAs (28, 29). Indeed Codium chloroplast tRNAG'WCC is as homologous to E. coli (65%), and even more homologous to phage T4 tRNAGIYUCC (73%), than to other chloroplast tRNAGIWCC species.

A very different level of homology is shown by the Codium chloroplast tRNALY"U*UU. This tRNA is quite homologous

TABLE I1 Percent sequence homology with Codium chloroplast tRNAs

The CCA at the 3' end is not included in the percent homology calculations. The tRNAs included in this table are from Refs. 8, 9, 25, and 42 and this paper.

Gly Lys Arg Met

Nicotiana chloroplast 65 85 76 73 Marchantia chloroplast 66 85 78 73 Triticum chloroplast 65 76 Zea mays chloroplast 76 76 Euglena chloroplast 55 79 73 65 Scenedesmus chloroplast 86 E. coli 65 63 65 69

17246 Characterization of Four Algal Chloroplast tRNAs

(85%) to the corresponding land plant chloroplast lysine tRNA isoacceptors, and is even 79% homologous to the chlo- roplast tRNALY8UUU of the unicellular alga Euglena (Table 11). The sequence homologies of the Codium chloroplast tRNAArg and tRNA,", while both fairly low, nevertheless fall between the level of homology shown by the glycine and lysine tRNAs. It is apparent from these homologies that some chlo- roplast tRNAs have been conserved to a much greater extent than others. Perhaps one factor involved in the greater con- servation of some tRNAs compared to others is that some tRNAs may have roles in addition to their role in protein synthesis, and that such tRNAs would tend to be more highly conserved. Roles for tRNAs in addition to protein synthesis are well known (30-33).

These four Codium chloroplast tRNAs hybridize to Codium chloroplast DNA and each has been restriction mapped to a specific site (Fig. 4). The location of these tRNA genes shows little resemblance to the location of the corresponding tRNA genes in both tobacco and M. polymorphia chloroplasts (Fig. 6). This agrees with the recent observation, based primarily upon protein coding genes, that the gene organization of the Codium chloroplast genome bears little resemblance to that of other chloroplast genomes characterized to date (13).

Codium Chloroplast tlPNA?'-This tRNA presents several interesting features. It contains m2G at position 10, a modi- fication characteristic of eukaryotic tRNA, and also found in archaebacterial and mitochondrial tRNA, but not found in eubacterial tRNA (25). This is the first example of this modification present at position 10 in a chloroplast tRNA. Interestingly, while this modification may be absent from a specific isoaccepting species of chloroplast tRNA, it may exist in the cytoplasmic counterpart from the same organism. Such is the case for the Scenedesmus obliquus and bean cytoplasmic initiator tRNAs which contain m2G at position 10 and the respective chloroplast initiator tRNAs from the same system do not (25). Similarly, m2G at position 10 is present in Euglena cytoplasmic tRNAPhe and absent in the same tRNA from Euglena chloroplasts (25). Since this Codium chloroplast tRNA," is the only known chloroplast tRNA to have the m2G modification at position 10, and there are 28 chloroplast tRNAs known (Ref. 25, and this paper), it appears that this modification is rarely present in chloroplast tRNAs. It would be most interesting to characterize the Codium m2G methyl- transferase involved to determine if it is a chloroplast-specific tRNA methyltransferase.

Another unusual feature of the Codium methionine elon- gator tRNA is the presence of a G at position 48. With few exceptions, position 48 is almost always occupied by a pyrim- idine and is therefore considered a semi-invariant residue (29). Normally, the semi-invariant pyrimidine at position 48 interacts with the semi-invariant purine at position 15 and is

H lOkb

FIG. 6. Comparison of the location of chloroplast tRNA and rRNA genes in Codium, tobacco, and the liverwort M. poly- morpha. The complete nucleotide sequences of the tobacco (8) and M. polymorph (9) chloroplasts are known. The locations of the Codium chloroplast tRNA and rRNA genes were determined by hybridization to restriction digests. The genomes are aligned with the 16 S rRNA.

one of several such interactions that assist in the stabilization of tRNA tertiary conformation (29). Significantly, Codium tRNA? maintains the purine at position 15. In view of the "deviation" at position 48, it is not known whether this residue can or does participate in a tertiary interaction with residue 15, or if perhaps there are specific compensatory features of this molecule that would obviate the need for the p ~ ' ~ : P y r ~ ' interaction.

Codium Chloroplast tRNALysU*UU-The sequences of three chloroplast tRNALY"UUU genes are known, from to- bacco (8), M. polymorpha (9), and Euglena (42). The Codium chloroplast tRNALYsU*UU is the first chloroplast lysine tRNA that has been determined. The tRNALY8UUU species from tobacco, M. polymorph, and Euglena chloroplasts all have been found to contain an unusual feature, they lack the top base pair of the anticodon stem. This feature is not present in the Codium chloroplast lysine tRNA which has a normal base pair A27:U43 at that site (Fig. 1).

The tendency toward replacement of specific bases with A or U residues in Codium chloroplast tRNAs is clearly evident in this lysine tRNA. Comparing, for example, the correspond- ing Codium and tobacco chloroplast tRNALY"UUU isoaccep- tors, the two tRNAs differ at 11 positions. In 10 of these positions Codium lysine tRNA has an A or U residue replacing a G or C residue found in tobacco lysine tRNA. The last change is A-U neutral; Codium lysine tRNA has U in place of an A in tobacco. There is not one site where the tobacco chloroplast lysine tRNA has an A or U residue in place of a G or C residue in Codium. Essentially the same pattern of unusually high A-U content is also present in the Codium chloroplast 4.5 S RNA (73% A + U) (14) and 5 S ribosomal RNA (72% A + U).3

All tRNALY8UUU species, from eubacteria, eukaryotes, ar- chaebacteria, and mitochondria, a total of 25 species, have a characteristic G residue at position 10 (25). This G residue is also present in the Euglena chloroplast tRNALY"UUU, but surprisingly, the other three chloroplast tRNALY"UUU spe- cies, from tobacco, M. polymorpha, and Codium, have an A at this site.

In terms of the modified base pattern of the Codium tRNALYsU*UU, residues qZ8 and D47 are especially informa- tive. Both of these modifications are found in eukaryotic tRNAs and are rarely found (D47) or absent (#*') from eubac- terial tRNAs (25, 28).

Codium Chloroplast tRNAArgICG-In addition to this Cod- ium chloroplast arginine tRNA, there are 7 chloroplast tRNAArgACG genes which have been determined (25). When these sequences are compared, a composite chloroplast argi- nine tRNA can be constructed for this isoacceptor (Fig. 7). This composite suggests that specific regions of the chloro- plast arginine tRNA are more highly conserved than others. For example, the nucleotides of the dihydrouridine stem and T$C stem regions are more conserved than the nucleotides of the anticodon stem region. Essentially the same pattern is also seen for the chloroplast methionine elongator tRNAs, where a composite based upon 9 chloroplast tRNAs can be constructed (data not shown).

Interestingly, Codium chloroplast tRNAArgICG contains the base pair U6:A'j7 in the acceptor stem, a base pair that is specifically absent in all of the 7 other chloroplast tRNAArgACG species. The Codium chloroplast tRNAArgICG is also different from the other 7 chloroplast arginine tRNA isoacceptors in 12 specific positions, where each of these 7 arginine tRNAs have a conserved residue and Codium has a different residue at that site. These positions for Codium, with the nucleotides found in the other 7 chloroplast tRNAArg

Characterization of Four Algal Chloroplast tRNAs 17247 . G C G . G C--70 .. .. .. 60

,, A I " A

c c G G G

U U C

55 I

30-6 "-40 G C

C A

U A

A C G

Chl. COMPOSITE

FIG. 7. Composite chloroplast tRNAArg. This tRNA sequence composite is based upon the sequences of 8 chloroplast arginine (ACG) tRNA or tRNA genes: Codium fragile (this paper), Euglena gracilis (25), Zea mays (25), Nicotina tabacum (25), Pelargonium zonale (25), Pisum satiuum (25), Spirodeka oligorhiza (25), and M. polymorhu (9).

species in parentheses, are: A7(G), A"(G), UZ5(C), $26(A), yF(C), AZs(G), U4'(C), A"(G), Um(C), A6'(U), A6'(G), and U7'(C).

Three modified bases present in Codium chloroplast tRNAA'gICG can be classified as typically eukaryotic in na- ture, $P6, @', and D47. The remaining modified residues, with the obvious exception of X37, occur widely in both eubacterial and eukaryotic tRNAs (28).

Codium Chloroplast tRNAGiyUCC-The most surprising feature of this tRNA is the presence of an unmodified U (U34), in the wobble position of the anticodon. This clear and un- ambiguous U was verified by thin layer chromatography in four different solvent systems. This is a very rare feature in tRNAs (25) and, with but a few exceptions (25), is limited to mitochondrial (and several other) systems in which tRNAs are capable of "extend.ed wobble" (34-36). This refers to the ability of specific tRNAs to read all four codons in a family box, instead of the usual two or three. This allows mitochon- dria to read the genetic code with fewer than the standard compliment of tRNAs. Do some chloroplast tRNAs also read the genetic code by extended wobble? Several lines of evidence suggest that this may indeed be the case. This evidence is as follows. (a) Several chloroplast tRNAs have an unmodified U in the wobble position of the anticodon. As discussed above, this is a very unusual feature in tRNAs in general, but is a characteristic of tRNAs which show extended wobble. In addition to the Codium chloroplast glycine tRNA reported here, several chloropllast leucine tRNAs (anticodon UAG) from soybean, bean, and spinach (37-39) and a partially sequenced threonine t:RNA from spinach chloroplast4 all have an unmodified U in the wobble position of the anticodon. It is significant too, that each of these tRNAs is responsible for reading within a codon family where extended wobble, should it occur, wouId not result in misreading the genetic code. All known mitochondrial tRNAs containing an unmodified U in the wobble position are restricted to codon families.

The codon reading properties of the leucine tRNA (anti- codon UAG) appear straightforward. This tRNA decodes the leucine CUN family and there is no other leucine tRNA

M. Kashdan and B. S . Dudock, unpublished results.

encoded in tobacco (8) or M. polynorpha chloroplasts (9) that can read the CUN family. It therefore appears that this leucine tRNA (anticodon UAG) is responsible for reading all four codons in the CUN family. Presumably this is done in a manner analogous to the mitochondrial tRNAs, where the unmodified U permits extended wobble to occur.

The situation with the Codium chloroplast glycine tRNA (anticodon UCC) is more puzzling. Due to the presence of the unmodified U in the wobble position it would be expected that this tRNA would read all four codons in the glycine GGN family. However, there is a second glycine tRNA species also present in Codium chloroplasts, as shown by tRNA amino- acylation assays5 Indeed we have recently isolated and se- quenced the gene for this glycine tRNA3 and found its anti- codon to be GCC. In addition, there are 2 glycine tRNA genes present in both tobacco (8) and liverwort (9) chloroplasts, and these are almost certainly analogous to those present in Codium chloroplasts. If the glycine tRNA (anticodon UCC) reads the genetic code by extended wobble why is a second, apparently redundant glycine tRNA species also present in chloroplasts? Essentially the same puzzling situation also occurs for the threonine tRNA, where a second, apparently redundant species of threonine tRNA is also present in chlo- roplasts. There is one example in mitochondria that may be analogous. In Aspergillis mitochondria 2 glycine tRNA genes have been found (40). One species has the "normal" UCC anticodon, and presumably reads the 4 glycine codons by extended wobble as do other mitochondrial glycine tRNAs. In addition, there is a second, seemingly redundant glycine tRNA gene also present, with the anticodon ACC (40). Alter- natively, the second species of glycine and threonine tRNA$ present in chloroplasts may not be redundant, and for reasons currently unknown the glycine and threonine tRNAs with the unmodified U in their wobble positions may not be capable of reading the genetic code by extended wobble in chloroplasts.

( b ) The complete nucleotide sequence of the tobacco chlo- roplast genome has been determined and found to encode only one species of alanine and one species of proline tRNA (8). While it is possible that another isoacceptor for both alanine and proline is encoded in the tobacco chloroplast genome but not yet found (perhaps split by multiple introns, etc.), this possibility is unlikely. Moreover, there is little evidence for importation of tRNAs into chloroplasts. In the case of proline tRNA this possibility has been explored. The proline tRNA from spinach chloroplast has been isolated and sequenced (41). Extensive chromatography on BD-cellulose and RPC-5 columns at neutral and acidic pH, using homolo- gous spinach chloroplast synthetase and E. coli synthetase failed to show a second proline i~oacceptor.~ Thus it appears most likely that for proline tRNA (and presumably for alanine tRNA as well) there may be only one isoacceptor in the chloroplast and, since all of the codons are used in chloroplast protein synthesis, it is likely that these chloroplast tRNAs read all four codons in their family box. Interestingly, the spinach chloroplast proline tRNA that was sequenced did not have an unmodified U in the wobble position (41). It had an unknown modified uridine designated U*. This suggests sev- eral possibilities, including the possibility that an unmodified U may not be a requirement for a tRNA to be capable of extended wobble, or perhaps that some chloroplast tRNAs may read the genetic code by the "two out of three" hypothesis of Lagerkvist (43) .

In summary, it is clear from the above discussion that there are a variety of aspects of the codon reading properties of chloroplast tRNAs that are not understood at present. These

M. A. Francis and B. S. Dudock, unpublished results.

17248 Characterization of Four

questions may be addressed by direct codon binding studies in homologous chloroplast systems with purified chloroplast tRNAs, chloroplast ribosomes, and chloroplast protein syn- thesis factors.

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1762

Characterization of Four Algal Chloroplast tRNAs 17249

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