Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

Abstract

Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

Keywords: Human RNA polymerase III; PTF; SNAPC; TFIIIA; TFIIIB; TFIIIC; activated transcription; basal transcription; enhancer; in vitro; promoter.

Figure 1. Schematic representation of the regulatory elements of human RNA polymerase III transcribed type 1 and type 2 genes. Nucleic acid nomenclature: R = G/A; Y = C/T; W = A/T. Designations of individual boxes are represented in appropriate color codes below type 1 and 2 gene schemes. Type 1 gene: 16 out of 17 human RNA5S genes retrieved at www.ncbi.nlm.nih.gov/nucleotide are identical; T84 within the C-box of RNA5S9 is substituted by a C. The sequence blocks of A- and C-boxes, as well as of the intermediate element (IE) in the human 5S gene are chosen by analogy to described sequence blocks in the X. laevis 5S RNA gene.^, The 5S gene A-box sequence lacks the conserved T in the first position of the tRNA consensus A box. The human D-box element sequence upstream of the transcription start site of the 5S gene was published. Type 2 genes: are representatively depicted by human tRNA consensus sequences that have been derived from published data.^- tRNA termination occurs at variable sites downstream of the B-box.

Figure 3. Schematic representation of human RNA polymerase III transcribed genes with regulatory elements 5′ and 3′ of the transcription start site (type 4 genes). Nucleic acid nomenclature: K = G/T; Y = C/T; R = G/A; S = G/C; W = A/T. Nucleotide sequences of individual presumably regulatory elements are shown for each gene. Designations of individual boxes are represented in appropriate color codes below type 4 gene schemes. Experimental proof for the functionality of these elements has been shown for the ATF/CRE element in the human 7SL and herpesvirus 4 EBER genes.^,,, Inter-species sequence conservation was shown for VTRNA1–1, -2, -3 and VTRNA2–1 regulatory elements. Vault distal elements comprise the shown sequences and two distinct additional further distal sequence blocks for each gene.^, PSE and STAF-binding sites in the human TRNAU1, RN7SL1 and 2, as well as the BCYRN1 gene were delineated by sequence homology with PSE and STAF-binding site consensus sequences (Fig. 2). TATA-like elements were proposed for the VTRNA1–1, -2, -3, the 7SL1 and 2 genes, EBER genes (ETAB) and the mouse analog of the BCYRN1 gene.^,, Sequences at similar positions in VTRNA2–1 and TRNAU1 genes are shown. A stimulatory influence of a SP1 binding site on in vitro transcription of the EBER2 gene was shown. An octamer element as a potential enhancer element was identified by sequence homology upstream of the BCYRN1 gene. Gene internal A and B boxes are appropriately depicted within the transcribed regions. Bases that differ from the tRNA consensus sequences (Fig. 1) for these elements are shown in black letters.

Figure 2. Schematic representation of regulatory elements of RNA polymerase III transcribed type 3 genes. The sequences and regulatory elements located 5′ of the transcription start site of the H1 (RPPH1), MRP (RMRP), U6ATAC (RNU6ATAC), U6–1 to U6–9 (RNU6–1, -2, -7, -8 and -9), 7SK (RN7SK), Y1 to Y5 (RNY-1, -3, -4 and -5) are depicted. Consensus sequences of the octamer element (red), STAF-binding site (blue), proximal sequence element (PSE; green) and TATA-like sequences (orange) are shown below. Nucleic acid nomenclature: B = T/C/G; D = A/T/G; H = A/C/T; V = C/A/G; K = G/T; M = A/C; S = G/C; W = A/T; Y = C/T; R = G/A; n = any of the four bases. Typical distances between the transcription start site (TSS) and regulatory boxes are indicated below the representation of the genes. The distances spanned by individual DNA elements, as well as those between the TSS and the TATA-like box and between the TATA-like box and the PSE are indicated by arrows and number of nucleotides (nt). The distal sequence element (DSE) is typically found 175 to 248 nt upstream of the TSS and composed of an octamer element and a STAF binding site. Octamer element and an inverted STAF-binding site are located closer to the transcription start site of the RPPH1 gene. The RMRP gene possesses an octamer element only at around -215 and the RNU6ATAC gene that comprises a potential STAF-binding site around -110. Sequences showing homology to inverted STAF-binding sites are located at -420 and -470, respectively, relative to the transcription start site of the RMRP gene (not shown). STAF (ZNF143) binding to the human U6 and H1 genes was demonstrated.^, The other STAF-binding sites have been deduced by sequence homology. PBP/PTF/SNAPc-binding to human U6 and 7SK genes has been demonstrated.^,, Potential PSEs in the other gene-regulatory sequences have been deduced by sequence homology. OCT-1 binding and stimulation of in vitro transcription of human 7SK and U6 genes was demonstrated.^, Deletion of the octamer element caused moderate effects on in vitro transcription of the RPPH1 gene.^,