UUA Codon Research Articles

Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony

The bldA gene encodes the only tRNA for the UUA codon that, although dispensable in genes important for primary vegetative growth of Streptomyces spp., is important in genes that serve a regulatory purpose in the differentiation. To investigate this role further, the spatial and temporal expression profiles of the bldA-regulated and unregulated genes within a Streptomyces colony were examined using modified genes for the green fluorescent protein ( gfp) as an expression-tag. A comparative study, based on computer-assisted quantitative analysis of the GFP fluorescence, revealed that the presence of TTA codons in gfp results in a temporal delay of translation and, consequently, changed the spatial pattern of the GFP expression within a colony, especially during early differentiation. The delay of GFP expression was undetectable at 60 h post-inoculation. These results provide the first extensive evidence that the bldA does indeed play a significant regulatory role during colony differentiation.

A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket.

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.

BldA dependence of undecylprodigiosin production in Streptomyces coelicolor A3(2) involves a pathway-specific regulatory cascade.

The production of the red-pigmented tripyrrole antibiotic undecylprodigiosin (Red) by Streptomyces coelicolor A3(2) depends on two pathway-specific regulatory genes, redD and redZ. RedD is homologous to several other proteins that regulate antibiotic production in streptomycetes; RedZ is a member of the response regulator family. redZ transcripts were detected during exponential growth and increased in amount during transition and stationary phases; transcription of redD was confined to the two latter stages of growth. Whereas mutation of redD had no effect on redZ transcription, transcription of redD was highly dependent on redZ, suggesting that RedZ is a transcriptional activator of redD. bldA, which encodes the only tRNA of S. coelicolor that can efficiently translate the rare leucine codon UUA, is required for Red production at higher phosphate concentrations. While the redD transcript contains no UUA codons, the redZ mRNA contains one. Transcription of redZ appeared to be unaffected in a bldA mutant; in contrast, redD transcription was undetectable, consistent with the translational dependence of redZ on bldA and the transcriptional dependence of redD on redZ. Red production in a bldA mutant was restored by multiple copies of redZ, presumably reflecting a low level of mistranslation of the redZ UUA codon, while multiple copies of redD had no effect, presumably a consequence of the severe dependence of redD transcription on RedZ. Transcription of redZ appears to be negatively autoregulated.

The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control.

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

The bldA-encoded tRNA is poorly expressed in the bldI mutant of Streptomyces coelicolor A3(2).

The bldA gene, encoding a leucyl tRNA recognizing the UUA codon, is expressed at significantly lower levels in the bldI mutant, Streptomyces coelicolor J703, than in the parent S. coelicolor A3(2). Expression of a TTA-containing reporter gene was reduced in the bldI mutant, as was the mature, 87 nucleotide, form of the bldA-encoded tRNA. This reduced level of the tRNA was also seen when the bldA gene was introduced on a high-copy-number plasmid into the bldI mutant, suggesting that maximal bldA expression may require a bldI-dependent promoter.

The Stringent Response, ppGpp and Antibiotic Production in Streptomyces coelicolor A3(2).

The correlation between ppGpp synthesis and the onset of antibiotic production in Streptomyces coelicolor A3(2) is reviewed. S. coelicolor A3(2) produces at least four antibiotics, two of which, actinorhodin and undecylprodigiosin (whose synthesis is determined by the act and red genes, respectively), are pigmented compounds. Conditions were established that gave dispersed and rapid exponential growth in liquid culture. Nitrogen limitation resulted in a short transition phase, during which a peak of ppGpp was observed, followed by stationary phase. Undecylprodigiosin production commenced in transition phase, while actinorhodin synthesis was detected 5∼6 hr later. Whereas the level of act and red biosynthetic transcripts increased markedly during transition phase, the level of the primary bldA transcript, which encodes the only tRNA of S. coelicolor A3(2) that can efficiently translate the rare leucine codon UUA and which is required for both actinorhodin and undecylprodigiosin production, decreased during early exponential phase; this decrease corresponded to an increased signal for the 5’ end of the mature tRNA, which then remained constant throughout growth. Nutritional shiftdown of exponentially growing cultures evoked immediate synthesis of ppGpp and a severe reduction in growth rate; transcription of act genes occurred shortly after, and was followed by actinorhodin production. However, there was no immediate stimulatory effect on red gene transcription, and undecylprodigiosin production did not occur. Addition of serine hydroxamate, a seryl tRNA synthetase inhibitor, to exponentially growing cultures led to ppGpp accumulation, but to lower levels than after nutritional shiftdown, and did not elicit antibiotic production. Thus only a partial correlation was observed between ppGpp synthesis and the transcription of antibiotic biosynthetic genes. S. coelicolor A3(2) DNA that hybridised to the Escherichia coli (p)ppGpp synthetase gene (relA) was cloned, but it did not encode a relA homologue, and polyclonal antibodies against RelA failed to reveal a homologue in S. coelicolor A3(2) cell extracts. A relA homologue of Serratia marcescens was isolated using the same approach, but failed to hybridise to S. coelicolor A3(2) DNA.

Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2)

Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.

Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated.

Production of actinorhodin, a polyketide antibiotic made by Streptomyces coelicolor A3(2), normally occurs only in stationary-phase cultures. S1 nuclease protection experiments showed that transcription of actII-ORF4, the activator gene required for expression of the biosynthetic structural genes, increased dramatically during the transition from exponential to stationary phase. The increase in actII-ORF4 expression was followed by transcription of the biosynthetic structural genes actIII and actVI-ORF1, and by the production of actinorhodin. The presence of actII-ORF4 on a multicopy plasmid resulted in enhanced levels of actII-ORF4 mRNA, and transcription of actIII and actinorhodin production during exponential growth, suggesting that actinorhodin synthesis in rapidly growing cultures is normally limited only by the availability of enough of the activator protein. bldA, which encodes a tRNA(Leu)UUA that is required for the efficient translation of a single UUA codon in the actII-ORF4 mRNA, was transcribed throughout growth. Moreover, translational fusions of the 5' end of actII-ORF4 that included the UUA codon to the ermE reporter gene demonstrated the presence of functional bldA tRNA in young, exponentially growing cultures and no increase in the efficiency of translation of UUA codons, relative to UUG codons, was observed during growth. The normal growth-phase-dependent production of actinorhodin in the liquid culture conditions used in these experiments appears to be mediated at the transcriptional level through activation of the actII-ORF4 promoter.

Presence and coding properties of 2′- O-methyl-5-carbamoylmethyluridine (ncm 5Um) in the wobble position of the anticodon of tRNA Leu (U*AA) from brewer's yeast

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA Leu(U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2′- O-methyl-5-carbamoylmethyluridine (ncm 5Um). The yeast tRNA Leu (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm 5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.

The use of a rare codon specifically during development?

A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNATTALeu, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic-production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.

Sequence of a new tRNA Leu(U ∗AA) from brewer's yeast

The nucleotide sequence of a new tRNA Leu(anticodon U ∗AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGUUGm 2GCac 4CGAGDGmGDCDAAGGCm 2GGCAGACmUU ∗AAm 1GAΨCUGUUGGACGGUUGUCCGm 5CGCGAGTΨCGm 1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA Leu(anticodon m 5CAA) and 63% homology with tRNA Leu(anticodon UAG), the two other known yeast tRNAs Leu.

Absence of acetohydroxy acid synthase III in Salmonella typhimurium is due to early termination of translation within the ilvl gene

The cryptic ilvlH locus of Salmonella typhimurium has genetic information for two distinct subunits of acetohydroxy acid synthase III. We show that the ilvH-encoded subunit is normally translated and the lack of activity is due to early termination of translation within the promoter-proximal ilvl gene. Analysis of the 5' region of the operon led to identification of the promoter and the amino-terminal part of ilvl. Expression of this gene in a mutant producing acetohydroxy acid synthase is due to a transversion which creates a UUA (leucine) codon in the place of a UGA (stop) codon present in position 12 of the wild-type coding region.

Phage Vectors that Allow Monitoring of Transcription of Secondary Metabolism Genes in Streptomyces

We describe a bacteriophage phi C31-based system that permits the transcriptional fusion of the convenient reporter gene xylE to chromosomally located promoters in Streptomyces hosts. Applicability of the system to genes for secondary metabolism is demonstrated in an experiment showing that transcription of genes for actinorhodin production in Streptomyces coelicolor A3(2) depends on a transfer RNA gene (bldA) for the rare UUA codon. Two other phi C31::xylE vectors are described that allow detection of promoter activity away from their natural location, either at single copy in a prophage or during lytic infections in plaques.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

The level of a transcript required for production of a Streptomyces coelicolor antibiotic is conditionally dependent on a tRNA gene

In Streptomyces coelicolor A3(2), bldA mutants are conditionally defective in aerial mycelium formation and fail to synthesize all four antibiotics produced by bldA+ strains. Previous studies showed that bldA specifies the tRNA for the rarely used leucine codon UUA. Here we describe experiments examining the abundance in a bldA mutant of a transcript involved in antibiotic production. With use of a bacteriophage-based integrative vector, a promotorless xylE reporter gene was inserted into a previously undescribed gene for an early step in biosynthesis of the red antibiotic undecylprodigiosin, located in the red gene cluster. With this transcriptional fusion present at unit copy number in the chromosome, xylE expression in a bldA+ strain was maximal late in growth in a liquid production medium and was virtually absent in a bldA mutant. On plates of a different medium, the bldA mutant was able to produce undecylprodigiosin and to express the red::xylE fusion, but both abilities were repressed by increasing the concentration of phosphate in the medium. These experiments showed that the undecylprodigiosin deficiency of bldA mutants cannot be accounted for by the presence of TTA codons in the red structural genes, but rather that bldA influences red gene mRNA abundance. In low-phosphate conditions, an alternative regulatory pathway can lead to red gene expression.

Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.

Does the 'non-coding' strand code?

The hypothesis that DNA strands complementary to the coding strand contain in phase coding sequences has been investigated. Statistical analysis of the 50 genes of bacteriophage T7 shows no significant correlation between patterns of codon usage on the coding and non-coding strands. In Bacillus and yeast genes the correlation observed is not different from that expected with random synonymous codon usage, while a high correlation seen in 52 E. coli genes can be explained in terms of an excess of RNY codons. A deficiency of UUA, CUA and UCA codons (complementary to termination) seems to be restricted to the E. coli genes, and may be due to low abundance of the relevant cognate tRNA species. Thus the analysis shows that the non-coding strand has the properties expected of a sequence complementary to a coding strand, with no indications that it encodes, or may have encoded, proteins.

The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem.

We have cloned and sequenced the wild-type and suppressor alleles of the S. pombe sup8 tRNA gene. The wild-type allele has a leucine UAA anticodon and the suppressor (sup8-e) carries the opal suppressor anticodon UCA. The gene has a 16 base pair intervening sequence that, in the RNA, is predicted to form a secondary structure which involves base pairing to the 5', rather than the usual 3' side of the 5' splice site. When incubated in Saccharomyces cerevisiae cell-free extracts both alleles are efficiently transcribed, the 5' leader and 3' trailer sequences are removed and CCA is added to the 3' processed end; however, the intervening sequence is not excised. This finding implies that the structural requirements of the splicing endonucleases in the two yeasts have diverged. No other tRNA genes with related sequences were detected in S. pombe DNA by hybridization, suggesting that other UUA isoacceptors may be structurally dissimilar to sup8 or that the UUA codon may be decoded by a UUG leucine isoacceptor.

Specificity of codon recognition by Escherichia coli tRNA Leu isoaccepting species determined by protein synthesis in vitro directed by phage RNA

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA 1 Leu and sequenced tRNA 3 Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA 3 Leu is highly preferred for the CUU codon; tRNA 4 Leu and tRNA 5 Leu both recognize UUA and UUG codons, with tRNA 4 Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX 4 (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX 4 leucine codon series is not paralleled in the UUX 4 leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.

Readinbg of the lysine codons in the MS 2 coat protein cistron during protein synthesis in vitro

The translation of the genetic information contained in the messenger RNA is based on the recognition between codon and anticodon. The first two positians of the codon are read by the anticodon strictly according to the rules of classic base pairing. Wowever, in the reading of the third codon position by the wobble nucleotide of the a~tic~dQ~ a greater degree of freedom must be postulated in order to allow for the discrepancy between the large number of codons in a degenerate code and the limited number of anticodons available for reading them. In his wobble hypothesis [I] Crick has laid down the restrictions which presumably govern the reading of the third codon position. Obviously, the reading of this position must be restricted in alI cases where there would otherwise be a mistake in the synthesized protein. For intance, in the phenyl~~ine~eu~~e codon group QJUU, UUC, UUA and UUG), an anticodon with Gin the wobble position cannot recognize the codons UUA and UUG because that would lead to the in~oduction of phenyl~an~e instead of leucine. We have introduced the term codon family to denote a group of four codons which all code for the same amino acid and we have asked ourselves if the rules of the wobble hypothesis apply as strictly here