UV Light‐induced Cross‐linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy‐terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry

Abstract

We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymidine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5'-monophosphate) and a dinucleotide (thymidylyl-[3'-->5']-2'-deoxyadenosine). The peptide picked was SPSYSPT (L-seryl-L-prolyl-L-seryl-L-tyrosyl-L-seryl-L-prolyl-L-threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.

Identification of RNA-associated peptides, iRAP, defines precise sites of protein-RNA interaction

“…Most amino acids can form photo-adducts with oligo-DNA or RNA sequences when single amino acids are added to the reaction, with Cys, Lys, Phe, Trp, Tyr, His, Glu and Asp being the most reactive amino acids towards DNA (Shetlar, 1980, Shetlar, Christensen et al, 1984, Shetlar, Home et al, 1984. Based on previous analyses (Bley et al, 2011, Connor et al, 1998, Doneanu et al, 2004, Maly et al, 1980, Williams & Konigsberg, 1991, Zwieb & Brimacombe, 1979, aromatic amino acids would also be expected to be among the most reactive with intact proteins, and this was found in our analyses. In previous analyses, positively charged (Lys, Arg) or polar amino acids (Asn, Cys, Gln, His, Ser, Thr, Tyr, Trp) were 1 0 largely implicated in interactions with nucleic acids, via either nitrogen bases or phosphate group (Ellis, Broom et al, 2007, Lejeune, Delsaux et al, 2005, Treger & Westhof, 2001 whereas we saw enrichment for hydrophobic amino acids (Ile, Leu, Val, Ala).…”

“…Despite this, we were able to identify UV-specific peptide modification with a mass of 324 Da, corresponding to the addition of a uridine monophosphate, without any losses of functional groups or atoms. It has been noted previously that in the photochemical reactions involving thymidine and tyrosine, the masses of the adducts formed were found to be equal to the sum of the masses of individual species (Connor, Falick et al, 1998).…”

DNA-Bound Peptide Radicals Generated through DNA-Mediated Electron Transport

“…Both peaks showed the mass of the corresponding nucleoside peptide adduct (dA′ m/z ) 717.7 [M + 23] + ; dT′ m/z ) 701.1 [M + 23] + ). It is noteworthy that these adducts occur only with adenosine and thymidine (36). This result, together with the different rise time of the tyrosine radical observed with DNA substrates 1-3 (Figure 5), establishes that Lys-Tyr-Lys binds preferentially to the AT alternating sequence.…”

Biochemical, biophysical, and proteomic approaches to study DNA helicases

“…50 Among these techniques, UV light is utilized to photochemically cross-link protein to nucleic acids, both of which absorb radiation in the 250-280 range. This approach has the advantage of behaving as a zero-length chemical linker [51][52][53] and femtosecond UV lasers have been recently utilized to increase the efficiency of the cross-linking reaction. 54,55 This cross-linking procedure may, however, lead to photodamage, which in turn, might affect the integrity and functionality of the components present in the complex.…”