Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex forma- tion is dependent on the DNA region extending from the 5’ end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcrip- tion complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kineti- cally dependent on the sequence 5’ to the gene region encoding the D-stem, and thermodynamically depend- ent on the gene region encoding the D-stem and -loop. Eukaryotic polymerase complex The intro-duction systems

Transcription of eukaryotic tRNA genes is dependent on the A- and B-Box internal control regions (ICRs) and the upstream transcription modulatory region. The B-Box ICR spans nucleotides 52 to 62 and directs the primary binding of transcription factor C as the first step in the formation of a transcription complex. The conservation of the sequence of the B-Box in all tRNA species reflects its importance in both the expression of the gene and the processing, structure and function of the gene product. In order to identify the nucleotides essential to the promoter function of the B-Box ICR, site-directed mutagenesis was used to generate all the possible single point mutations at positions 52 to 58, 61 and 62 of a Drosophila melanogaster tRNA(Arg) gene. The effect of these mutations on gene transcription was evaluated using in vitro transcription and template exclusion competition assays. Optimal activity was displayed by the wild type tDNA(Arg) B-Box sequence but several other sequences supported in vitro transcription at wild type levels. The majority of mutants, however, showed lower efficiency in the in vitro transcription assay. Of the single point mutations, those at positions 53, 55, and 56 had a critical effect on gene function in Drosophila and HeLa transcription extracts and transcription factor interaction most likely requires base contacts at these positions. Since the effect of several of the point mutations cannot be explained in terms of possible major or minor groove contributions the possibility is raised that local DNA geometry also is an important determinant in specifying B-Box function.

Linker-scanning (LS) mutations were constructed spanning the length of the Drosophila melanogaster 5S RNA gene. In vitro transcription analysis of the LS 5S DNAs revealed five transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants of this upstream promoter region were located between coordinates -39 and -26 (-30 region), but important sequences extended to the transcription start site at position 1. Since mutations in the upstream promoter did not alter the ability of 5S DNA to sequester transcription factors into a stable transcription complex, it appears that this control region involved the interaction of RNA polymerase III. Active 5S DNA transcription additionally required the four intragenic control regions (ICRs) located between coordinates 3 and 18 (ICR I), 37 and 44 (ICR II), 48 and 61 (ICR III), and 78 and 98 (ICR IV). LS mutations in each ICR decreased the ability of 5S DNA to sequester transcription factors. ICR III, ICR IV, and the spacer sequence between were similar in sequence and position to the determinant elements of the multipartite ICR of Xenopus 5S DNA. The importance of ICR III and ICR IV in transcription initiation and in sequestering transcription factors suggests the presence of an activity in D. melanogaster similar to transcription factor TFIIIA of Xenopus laevis and HeLa cells. Transcription initiation of Drosophila 5S DNA was not eliminated by LS mutations in the spacer region even though these mutations reduced the ability of the TFIIIA-like activity to bind. The previously unidentified control regions ICR I and ICR II appear to be important for the interaction of a transcription factor activity, or multiple-factor activities, distinct from the TFIIIA-like activity. The interaction of this activity with ICR I directed the selection of the transcription start site.

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

Three tRNA5Asn genes have been subcloned from a tRNA gene cluster isolated from the cytogenetic locus 42A of Drosophila melanogaster. The three tRNAAsn genes, contained on plasmids pAsn6, pAsn7, and pAsn8, have identical mature tRNA coding regions but have different 5'- and 3'-flanking sequences. In vitro transcription in Drosophila Schneider S3 cell-free extracts showed the tRNAAsn genes had different transcription efficiencies. pAsn8 had a transcription efficiency of approximately 8 transcripts/gene/h, whereas pAsn6 was a less active template at 5 transcripts/gene/h. pAsn7 was the poorest template at 1.5 transcripts/gene/h. Exchanging 5'-flanking regions of the tRNAAsn genes showed that the differences in transcription efficiencies were attributable to the corresponding 5'-flanking region. Transcription of each of the tRNAAsn genes revealed a different optimum for KC1 concentration for each template which also was directly attributable to the corresponding 5'-flanking region. The "salt effect" is not related to the ability of the three tRNAAsn genes to sequester transcription factors as determined using the stable complex competition assay. Rather, this effect appears to be due to the ability of the respective 5'-flanking regions to interact with RNA polymerase III. The poorest transcription template, pAsn7, was a better competitor in the stable complex formation assay than either pAsn8 or pAsn6. We conclude that the pAsn7 stable complex binds and functionally arrests RNA polymerase III in the initiation reaction.

We have examined the effects of various nucleotide substitutions in a Drosophila tRNAArg gene on in vitro transcription and stable transcription complex formation in Drosophila KcO and HeLa cell extracts. Substitutions in positions encoding the invariant G18 and G19 residues resulted in decreased transcription, however, the moderate decreases indicate that these nucleotides are not obligatory promoter recognition sites. An A21 to C21 mutation had no effect on transcription levels using homologous extract however, this mutant displayed decreased transcriptional abilities in HeLa cell extract. Nucleotide substitutions within the sequence encoding the anticodon led to a decrease in the transcription activity but not in the ability to form a stable transcription complex.

The availability of cloned tRNA genes and a variety of eukaryotic in vitro transcription systems allowed rapid progress during the past few years in the characterization of signals in the DNA-controlling gene transcription and in the processing of the precurser RNAs formed. This will be the subject matter discussed in this review.

Publisher Summary This chapter focuses on characterizing the function of Oβ-phosphoseryl-tRNA. The approaches that are used to determine whether Oβ-phosphoseryl-tRNA (SerP-tRNA) has a translational role in the synthesis of structural phosphoproteins include determining the structure of tRNASerP and casein mRNAs by nucleic acid sequencing methods. The sequence data predicts that SerP-tRNA does not incorporate SerP into casein as part of the mechanism for casein phosphorylation. SerP-tRNAs possess a tryptophan anticodon but respond, in vitro, to the termination codon UGA. Also a study of the involvement of SerP-tRNA in in vitro translation systems has demonstrated that this tRNA can suppress the UGA termination codon of β-globin mRNA in a reticulocyte lysate producing an identifiable globin readthrough product. A direct approach to determine a role for SerP-tRNA in phosphoprotein synthesis is to examine the codon usage for SerP residues in such proteins. For recognizing direct involvement of SerP-tRNA in translation, a mechanism in which the UGA could be accepted out of phase is essential.

Transcription of isolated repeat units of D. melanogaster 5S DNA in a Drosophila KcO cell extract revealed three types of template activities. 5SI DNA encodes the known 5S rRNA of D. melanogaster and has a relatively high transcription efficiency. 5SII DNA is identical to 5SI DNA except for a two-nucleotide deletion at 5S rRNA positions 28 and 29; the efficiency of transcription is approximately 40% that of 5SI DNA and because of the deletion, the primary transcript is two nucleotides shorter. 5SIII DNA does not support in vitro transcription (less than 2% 5SI DNA), but has the same sequence as 5SI DNA except for a single G to A transition at position 86. This is the first reported point-mutation in a 5S RNA gene resulting in loss of transcription function. Of approximately 23 5S rRNA gene copies in a cloned 5S DNA sub-cluster (p12D1) 19 appear to be of the transcriptionally inactive 5SIII DNA type.

We have examined the 5'-and 3'-flanking sequence requirements for the wild type transcription properties of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.

Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex formation is dependent on the DNA region extending from the 5' end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcription complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kinetically dependent on the sequence 5' to the gene region encoding the D-stem, and thermodynamically dependent on the gene region encoding the D-stem and -loop.

The regions of a Drosophila tRNAArg gene responsible for the "wild type" in vitro transcription level were determined by a transcription-competition assay. Cell-free transcription extracts programmed with 5' and 3' deletion mutants of the tRNAArg gene were used to quantitate the efficiency of transcription and to measure the ability of these DNAs to compete for transcription factors compared to the wild type tRNAArg gene. The results show that those portions of the gene which code for the D-stem/D-loop and T-stem/T-loop of the tRNA product are the regions responsible for competitive ability. These regions were previously shown to contain the intragenic control sequences for eukaryotic tRNA gene transcription and are respectively referred to as the D- and T-control regions. The presence of both the D- and T-control regions is essential for maximum competitive strength. The 5'-flanking and 5' stem regions adjacent to the D-control region have a function in the competitive ability of the D-control region while the 3'-flanking and the 3' stem regions adjacent to the T-control region have a function in the competitive ability of the T-control region. These results are consistent with a model for promotion of tRNA gene transcription that involves recognition by transcription factors of the two control regions. Optimal binding of the transcription factors is dependent upon sequences adjacent to and flanking the intragenic control regions.

The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.