Table 2 Oligonucleotides used in the study’
From: Mycobacterial HelD connects RNA polymerase recycling with transcription initiation
Oligonucleotides | Sequence 5 → 3′ | Description |
|---|---|---|
TK1 | CAGGAGAACCTGTACTTCCAGGGCATGGCAGCGACAAAGGCAAG | Primers for cloning of the Msm σA gene (MSMEG_2758) into pET28-MBP-TEV |
TK2 | TGCCCTGGAAGTACAGGTTTTCTTCTCGAGCTAGTCCAGGTAGTCGC | |
TK71 | GCTTGACAAAAGTGTTAAATTGTGCTATACT | |
TK72 | CAATTTAACACTTTTGTCAAGC | this study |
3286/ HelD-FLAG_ra_F | AACTTCTCTAGACTCAGTTCGCACACGCCTG | Primers for the right homology arm of HelD (MSMEG_2174) |
3287/ HelD-FLAG_ra_R | TACGAATTCGAGCTCGGTACCCGGGGATCCCTCGACCCGTTCGTCGAC | |
3290/ HelD-FLAG_la_F | CGTTGTAAAACGACGGCCAGTGCCAAGCTTCGTGGGAGACCTGGCGCAG | Primers for the left homology arm of HelD (MSMEG_2174) |
3291/ HelD-FLAG_la_R | CGTTAACCTGCAGCTACTTGTCGTCGTCGTCCTTGTAG | |
3292/ hygro-HelD-FLAG_F | GACGACAAGTAGCTGCAGGTTAACGAAATCAATC | Primers for the hygromycin resistance |
3293/ hygro-HelD-FLAG_R | TGTGCGAACTGAGTCTAGAGAAGTTATCCCGGG | |
3771/ RbpA_No_tag_F | CGAGGAAAACCTGTACTTCCAGGGTATGGCTGATCGTGTCCTGCGG | Primers for cloning of Msm rbpA (MSMEG_3858) into pET302/NT-His. |
3772/ RbpA_No_tag_R | CTTTCGGGCTTTGTTAGCAGCCGGATCCTTAGCTTCCGGTTCCGCGCCG | |
4394/nt-strand_OC | GCTTGACAAAAGTGTTAAATTGTGCTATACTGGGAGCCGTCACGGATGCG | Non-template strand to form open double-stranded DNA resembling transcription bubble30. |
4395/t-strand_OC | CGCATCCGTGAGTCGAGGGTAATAAGCACAATTTAACACTTTTGTCAAGC | Template strand to form open double-stranded DNA resembling transcription bubble |
4396/nt-strand_CC | GCTTGACAAAAGTGTTAAATTGTGCTATACT | Non-template strand DNA to form closed double-stranded DNA resembling the closed promoter complex30 |
4397/t-strand_CC | AGCACAATTTAACACTTTTGTCAAGC | Template strand DNA to form closed double-stranded DNA resembling the closed promoter complex |
JD1/HelD sigA-INT_F | TCGACCTGTCTGCGGTTACCATGCGTATCGACGCTGAAACCGCTAAAGCGGCTCG TGACGAAGCT | Forward primer for mutagenesis of HelD to create LK_4162 (mutant of σA interaction interface) (MSMEG_2174 - D310A, W322 A, Y347D, W361A, H368A) |
JD2/HelD sigA-INT_R | CATTTTTTCCCAAGCGGCTTTGTCGTCACGGGTCAGCGCACCACGACCGATACG AGCATCAGCACGTTCGGTAACAACATCGGTAACAACGTCAAC | Reverse primer for mutagenesis of HelD to create LK_4162 (mutant of σA interaction interface) (MSMEG_2174 - D310A, W322A, Y347D, W361A, H368A) |
JD3/HelD sigA-HYDRO_F | CGTTGTTGTTGACAGCGCTCAGGAACTGTCTGAAAT | Forward primer for mutagenesis of HelD to create LK_4163 (mutant - ATP hydrolysis) (MSMEG_2174 - E529S, Q558N) |
JD4/HelD sigA-HYDRO_R | CGGAGAACGACGGTTAGCCAGGTCGCCAA | Reverse primer for mutagenesis of HelD to create LK_4163 (mutant - ATP hydrolysis) (MSMEG_2174 - E529S, Q558N) |
JD5/HelD sigA-BIND_F | TCCGGGTACCGGTAAAGAAGTTGTTGCTCTGCA | Primer for mutagenesis of HelD to create LK_4164 (mutant - ATP binding) (MSMEG_2174 – T206E) |
