A rho-dependent termination site in the gene coding for tyrosine tRNA su3 of Escherichia coli

Abstract

A set of partially overlapping DNA restriction fragments that support promoter-dependent transcription of the tRNATyr1 gene of Escherichia coli has been used to study site-specific termination in vitro. Transcription termination occurs at a specific site 224-226 nucleotides beyond the end of the structural gene and is completely dependent on rho-factor. Certain features of this site suggest differences from other termination sites previously studied. A role for specific sequence recognition is suggested.

Fig. 1

DNA restriction fragments used as templates for promoter-dependent transcription of the tyrosine tRNA su₃ ( ${\text{tRNA}}_{\text{1}}^{\text{Tyr}}$) gene. The isolation of Cla and Bla from the ‘singlet’ transducing phage, Φ80psu₃⁺³² has been described previously. The HpaII fragment is obtained by digestion of Bla with HpaII and purification by preparative polyacrylamide gel electrophoresis,. The approximate size of the fragments in base pairs is given in parentheses,. The 85 nucleotides coding for the mature tRNA sequence are indicated by the hatched box. These are preceded by 41 nucleotides coding for the 5′ end of the primary transcript or precursor molecule. The end of the promoter and the startpoint of transcription is marked (P). The ρ-dependent termination site is marked (T), and a potential downstream termination site is marked (T′). The 178-base pair repeat unit (see Fig. 4) is indicated by overhead brackets. Promoter-dependent transcription ( ) in the presence and absence of ρ factor is indicated.

Fig. 2

Effect of ρ factor on transcription from various templates carrying the ${\text{tRNA}}_{\text{1}}^{\text{Tyr}}$ gene. Standard conditions for RNA synthesis were 40 mM Tris-HCl pH 7.9, 10 mM MgCl₂, 10 mM 2-mercaptoethanol, 0.1 mM EDTA Na₃ and 15% glycerol in a final volume of 10 μl. Nucleotides were added to a final concentration of 500 μM CpC, 500 μM GTP, 15 μM each ATP, CTP and UTP. [α-³²P] UTP as the labelled triphosphate had a specific activity of 15,000 c.p.m. pmol⁻¹ (New England Nuclear). The dinucleoside monophosphate CpC was added to stimulate initiation of transcription. E. coli RNA polymerase (RNA nucleotidyl-transferase EC 2.7.7.6.) was prepared according to Burgess and was present at 1.5 pmol per 10 μl. Termination factor ρ (prepared according to Roberts3) was present as indicated, and the DNA fragments were added as follows: lane 1, 0.15 pmol Cla; lane 2, 0.15 pmol Cla and 0.25 pmol ρ; lane 3, 0.06 pmol HpaII; lane 4, 0.06 pmol HpaII and 0.25 pmol ρ; lane 5, 0.08 pmol Bla; lane 6, 0.08 pmol Bla and 0.25 pmol ρ; lane 7, 0.08 pmol Bla and 0.2 pmol ρ (a gift from Dennis Kleid); lane 8, 0.08 pmol Bla and 0.2 pmol ρ (a gift from Elizabeth Bikoff). Incubation was for 60 min at 37 °C. RNA synthesis was stopped by the addition of 50 μl electrophoresis loading buffer (7 M urea, 0.1% SDS, 90 mM Tris base, 90 mM boric acid, 4 mM EDTA Na₂, and the dye markers xylene cyanol and bromophenol blue), and heating for 2 min to 100 °C with subsequent quick cooling on ice. The RNA products were separated on a 4.5% polyacrylamide slab gel (20 × 20 × 0.2 cm) containing 7 M urea (acrylamide: N,N′ methylene bis acrylamide, 20:1). Electrophoresis was performed in TBE buffer (90 mM Tris base, 90 mM boric acid, 4 mM EDTA Na₂) at 50–70 mA until the bromophenol blue marker reached the bottom of the gel. M1 and M2 indicate the positions of the dye markers, bromophenol blue and xylene cyanol, respectively. The approximate size of the major transcription products (no. of nucleotides) is indicated in the left margin. The minor band which migrates just ahead of the ρ-dependent product (lanes 3–8) has not been analysed but is visually estimated to contain less than a few per cent of the total counts incorporated.

Fig. 3

a, Effect of salt concentration on ρ-dependent termination. RNA polymerase (3.2 pmol) was preincubated with Bla (0.45 pmol) in the presence of CpC and GTP (500 mM each) in standard conditions (see Fig. 2) in a total volume of 35 μl for 10 min at 37 °C. Aliquots of 7 μl were withdrawn, and the remaining triphosphates were added to a final concentration of 15 μM along with ρ (0.25 pmol per assay) and KCl as indicated. The mixtures (final volume 10 μl) were further incubated for 50 min at 37 °C. This preincubation and the prior formation of initiation complexes are necessary because of the salt-sensitivity of the tRNA^tyrT promoter (see ref. 11). Transcription was stopped, and RNA products were analysed on a 4% polyacrylamide gel as described in Fig. 2. Lane 1, −ρ, −KCl; lane 2, + ρ, −KCl; lane3, +ρ, 50mM KCl; lane4, +ρ, 100mM KCl; lane 5, +ρ, 150mM KCl. b, effect of ρ factor on promoter-dependent initiation of transcription at different polymerase: DNA ratios. Varying amounts of RNA polymerase were incubated in standard conditions with the Cla fragment (0.1 pmol per 10 μl reaction) in the presence or absence of ρ factor (0.2 pmol per 10 μl). Incubation was 45 min at 37 °C, and the RNA products were analysed on a 5% polyacrylamide gel (see Fig. 2). Incorporation of radioactivity into the main product was measured by Cerenkov counting of the excised gel band. The RNA polymerase: DNA ratios for each reaction and the amount of radioactivity in the excised band were as follows: lane 1, 1.8:1, + ρ (1,388 c.p.m.); lane 2, 1.8:1, −ρ (1,400c.p.m.); lane 3, 3.7:1, +ρ (3,841 c.p.m.); lane 4, 3.7:1, −ρ (2,159 c.p.m.); lane 5, 7.5:1, +ρ (7,611 c.p.m.); lane 6, 7.5:1, −ρ (7,548 c.p.m.); lane 7, 15:1, +ρ (5,692 c.p.m.); lane 8, 15:1, −ρ (7,969 c.p.m.).

Fig. 4

Sequence of the distal region of the ${\text{tRNA}}_{\text{1}}^{\text{Tyr}}$ gene. The DNA strand having the same sequence as the tRNA is arranged to facilitate comparison of the 178-base pair repeating units. The first base shown (42) corresponds to the first base in the mature tRNA (the 41 nucleotides corresponding to the 5′ portion of the primary transcript, or precursor,, are not shown). The sequences corresponding to the mature tRNA are indicated by an overhead bracket, and that portion of this sequence which appears at the beginning of each repeat unit by a dashed bracket (see text and ref. 19). Positions in one repeat which differ from the others are marked by an asterisk. The location of the ρ-dependent termination of transcription is marked (T) (see text and Fig. 5). The downstream termini of the three restriction fragments used as templates for transcription in this study are also indicated (see Fig. 1).

Fig. 5

Analysis of the 3′-terminal oligonucleotides of the promoter-dependent, ρ-dependent, transcript. The 360-nucIeotide transcript from the HpaII reaction (see Fig. 2) was prepared and isolated as described in legend Fig. 2 with the following preparative modifications: total reaction volume, 30μ;l HpaII restriction fragment, 0.3 pmol; RNA polymerase, 5.0 pmol; p factor, 0.6 pmol; [α-³²P]UTP at 15 μM with a specific activity of 1–1.5 × 10⁵ c.p.m. pmol⁻¹, GTP, CTP, and ATP each at 500 μM. The radioactive band was excised from the gel and disrupted in a 5 ml syringe. The RNA was eluted with 1 M NaCl and recovered by ethanol precipitation as described previously. The RNA was digested with T1 ribonuclease and passed over a column of DEAE-cellulose as described previously. Oligonucleotides containing a 3′-terminaI phosphate were eluted with solvent A (0.05 M morpholinium chloride, 0.1 M MgCl₂, 1.0 M NaCl, 20% dimethylsulphoxide, pH 8.7). Oligonucleotides which contain a nucleoside at their 3′ end, and thus should be derived from the 3′ terminus of the transcript, were subsequently eluted from the column by simple displacement with solvent A containing 0.1 M sorbitol. The 3′-oligonucleotides were desalted by dialysis against distilled water, concentrated under reduced pressure at 35 °C, and fractionated in the first dimension by electrophpresis on cellogel in 8 M urea at pH 3.5 and in the second dimension by ascending thin layer chromotography on plates of DEAE-cellulose (9:1, cellulose :DEAE-ceilulose, 40 × 20 cm) using homochromatography solvent B–. The radioactive spots were eluted, quantified by Cerenkov counting, and subjected to further analysis by pancreatic ribonuclease digestion using standard techniques–. The schematic diagram next to the autoradiogram shows the sequence deduced for each spot, the single base differences deduced on the basis of mobility shifts between the spots, and the per cent of the total radioactivity found in each spot.