The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit.

The 39S ribosomal RNA (rRNA) precursor has been isolated from Acanthamoeba castellanii. In vitro capping of the isolated RNA verified that it is the primary transcript and identified the 5' nucleotide as pppA. The position of the 5' coding nucleotide on the rRNA repeat unit sequence was identified using Northern blot, R-loop, and S1 nuclease mapping techniques. Dinucleotide priming of an in vitro transcription system stalled because of low initiating nucleotide concentration revealed that ApA maximally stimulates initiation of transcription. All of these results show that the underlined A in the sequence 5'-TATATATAAAGGGAC (RNA-like strand) coincides with the 5' nucleotide of the primary transcript. This identification is compatible with in vitro transcription experiments mapping the promoter for this transcription unit. The initiation sequences of rRNA genes from 14 species are compared, and a weak consensus for the initiator derived: [Formula; see text].

Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii.

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.

Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I.

Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent.

RNA polymerase I binding to the eukaryotic ribosomal RNA gene promoter-transcription initiation factor (TIF) complex was examined by in vitro transcription and footprinting of a series of spacer mutants. Polymerase binds efficiently to the TIF-promoter complex independently of the DNA sequence in the polymerase interaction region and initiates transcription a fixed distance downstream of the TIF binding site on AT-rich templates. Methidiumpropyl-EDTA.FE(II) footprinting confirms minimal contacts between polymerase and DNA. We infer that polymerase is directed to the promoter by a DNA sequence-independent mechanism, solely by protein-protein contacts with TIF. An initiation step subsequent to binding requires special sequence characteristics in the transcription start site region.

Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I.

The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from the transcription start site, protecting a region extending from around -14 to -67 on the coding strand, and -12 to -69 on the noncoding strand. The protein that binds to DNA within this region can be competed out by using wild-type promoters but not by using mutants which do not stably bind the factor. RNA polymerase I can form a stable complex in the presence of DNA and transcription factor, allowing footprinting of the complete transcription initiation complex. RNA polymerase I extends the protected region obtained with TIF alone to around +18 on the coding strand, and to +20 on the noncoding strand. This region is not protected by polymerase I in the absence of TIF. The close apposition of the regions protected by TIF and polymerase provides evidence that accurate transcription of the ribosomal gene may be achieved through protein-protein contacts as well as through DNA-protein interactions.

The ribosomal RNA promoter of Acanthamoeba castellanii determined by transcription in a cell-free system.

The DNA sequences required for faithful initiation of ribosomal RNA transcription were determined. BAL-31 digestion was used to modify the rDNA template by introducing deletions from its 3'- and 5'-ends. The resulting mutant DNAs were tested for template activity individually or in competition with wild type utilizing an in vitro transcription system from Acanthamoeba castellanii. The results identify the sequence extending from -31 to +8 to be absolutely required for transcription. In addition; when the region between -47 and -32 is left intact, transcription is augmented.

Ribosomal RNA transcription: proteins and DNA sequences involved in preinitiation complex formation.

An in vitro transcription system consisting of partially purified transcription initiation factor(s) and purified RNA polymerase I from Acanthamoeba castellanii was used to study the mechanism of faithful initiation of ribosomal RNA transcription. Formation of a preinitiation complex between one or several auxiliary transcription proteins and the DNA template in the absence of RNA polymerase I was demonstrated. A series of 3'- and 5'-deletion mutants of the template was used in prebinding competition experiments and provided evidence for three distinct functional regions of the promoter: core motif A interacts with the transcription initiation factor(s) and is required for faithful transcription; the start motif is required for transcription, but it can be deleted without affecting the binding of transcription initiation factor(s); and motif B stabilizes preinitiation complex formation (in addition to core motif A), but it is dispensable for faithful initiation of transcription.

Faithful initiation of ribosomal RNA transcription from cloned DNA by purified RNA polymerase I.

A faithful transcription system for ribosomal RNA genes has been developed by using components from the small free-living amoeba Acanthamoeba castellanii. The system utilizes protein-free recombinant DNA as a template and in addition requires a crude cell-free extract containing RNA polymerase I and a transcription initiation factor (TIF-I). The transcript is initiated at the same position as the in vivo precursor ribosomal RNA: templates truncated at various sites downstream of the transcription start site give rise to only the predicted runoff RNA transcripts, and the runoff transcript produced has a 5'-terminus identical with the 5'-terminus of the isolated ribosomal RNA precursor. Faithful initiation can be elicited by the DNA sequence extending from -55 to +19 in the template. Subclones containing this sequence yield only the predicted runoff RNAs regardless of the orientation of this fragment in the cloning vector DNA; thus, only the in vivo sense strand of the template is specifically transcribed in the in vitro system. The system is specific for the RNA polymerase responsible for the transcription of ribosomal RNA genes in vivo. Faithful transcription, like RNA polymerase I from Acanthamoeba, is insensitive to alpha-amanitin inhibition, and transcription is greatly stimulated by highly purified RNA polymerase I but not by RNA polymerases II or III. Conditions for optimal transcription were determined.