Preferential interaction of the mRNA proofreading factor TFIIS zinc ribbon with rU.dA base pairs correlates with its function.
The transcriptional factor TFIIS helps overcome elongation barriers and enhances proofreading by RNA polymerase II. These TFIIS functions may be modulated by the TFIIS zinc ribbon domain through interactions with nucleic acids in the elongation complex. Within this zinc ribbon domain, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have been shown to be critical for its function by mutant analysis. The sequence Asp261-Glu262 has been suggested to participate in metal binding within the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of electrostatic and stacking interactions. The interaction of the indole side chain of the tryptophan residue with nucleic acid bases is demonstrated by a characteristic and reversible decrease in the zinc ribbon fluorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM and no interaction at 500 mM NaCl), suggesting that the tryptophan stacking with nucleic acid base accompanies electrostatic contacts. The oligonucleotide-zinc ribbon interactions exhibit small but significant base preferences, as shown by the dependence of Keq on base composition, with decreasing Keq in the order U > T > A > C >> G. Within the variety of homopolymeric single- and double-stranded deoxy- and ribooligonucleotides, the oligonucleotide rU12-18.dA20 exhibited a 2-6-fold binding preference relative to other oligonucleotides. This preferential binding of the zinc ribbon to sequences composed of rU.dA base pairs, which are generally associated with elongation blocks, may help in overcoming elongation barriers. Since the mRNA proofreading and enhancement of elongation involve cleavage of ribonucleotide of the mismatched pair and the weakly paired rU.dA nucleotides, but not the stably paired rC.dG nucleotides, we propose that the Arg276-Trp277 sequence in the TFIIS zinc ribbon may serve as a scanner connected to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a criterion of determining their subsequent removal. The striking similarity in preference for mismatched and weakly paired nucleotides for binding and for excision suggests a functional relationship between binding and cleavage reactions.
Department of Biochemistry, The University of Chicago, Illinois 60637, USA.
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Yoon
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Sitikov
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Jeon
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Agarwal
D001120:Arginine / Q000378:metabolism; D001482:Base Composition; D005454:Fluorescence Polarization; D046911:Macromolecular Substances; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011072:Poly U / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D014364:Tryptophan / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010445:Peptide Elongation Factors; D011742:Pyrimidine Nucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C033446:oligo(dA)-poly(rU); C018648:transcription factor S-II; D011072:Poly U; D014364:Tryptophan; D001120:Arginine; D012319:RNA Polymerase II; D015032:Zinc