Protein Environment of the Sense Codon of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking to Oligoribonucleotide Derivatives

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA^Phe, which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA^Phe and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.     

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.     

The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU

Three mRNA analogs--derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position--were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA(Phe) was used, while tRNA(Val) was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions -3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with soft UV light (lambda > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions -1 to +3 binds to G1207, while that in positions -2 or -3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the -3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the -3 position, this crosslinking was not observed in the absence of tRNA.     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.     

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA^Phe was used, while tRNA^Val was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light ( > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.     

The C-terminal fragment of ribosomal protein S15 is located in the decoding site of the human ribosome

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors the mRNA codon at the decoding site and the downstream triplets. The S15 fragment juxtaposed in the human ribosome to mRNA nucleotides +4 to +12 relative to the first nucleotide of the P-site codon was determined. S15 was modified using a set of mRNA analogs containing the triplet UUU/UUC at the 5′ end and a perfluorophenyl azide-carrying uridine at various positions downstream of this triplet. The mRNA analogs were positioned on the ribosome with the use of tRNA^Phe, cognate to the UUU/UUC triplet, targeted to the P site. Modified S15 was isolated from complexes of 80S ribosomes with tRNA^Phe and the mRNA analogs after irradiation with mild UV light and hydrolyzed with cyanogen bromide, cleaving the polypeptide chain after Met residues. Analysis of the modified oligopeptides resulting from hydrolysis demonstrated that the crosslinking site was in C-terminal fragment 111–145 of S15 in all cases, suggesting the involvement of this fragment in the decoding site of the eukaryotic ribosome.     

Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, UCC-recognizing tRNA(Phe) was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 or nucleotide A1825 in helix 44 close to the 3' end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem-loop segment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in various organisms.     

Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.     

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: a crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.     

The Template Region 5′ of the E-Site Codon Is Close to S26 in the Human 80S Ribosome

The environment of the template sequence 5 of the E-site codon on the 80S ribosome was studied with nonaribonucleotide or dodecaribonucleotide derivatives containing Phe codon UUU at the 3 end and a perfluoroarylazido group at the first or third nucleotide. A photoreactive group was linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which is cognate to the UUU codon and directs it to the P site, bringing a modified nucleotide in position –4 to –9 relative to the first nucleotide of the P-site codon. Upon irradiation of ribosome complexes with tRNA^Phe and the mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins. The major target of modification was S26 in all cases. In addition, S3 was modified to a low extent when the reactive nucleotide was in position –4 and S14 was in position –6. In the absence of tRNA, all mRNA analogs modified S3.     

Protein S3 fragments neighboring mRNA during elongation and termination of translation on the human ribosome

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNA^Phe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.     

C-terminal fragment of ribosomal protein S15 is located at the decoding site of the human ribosome

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors mRNA codon at the decoding site and the downstream triplets. In this study we determined S15 protein fragments located close to mRNA positions +4 to +12 with respect to the first nucleotide of the P site codon on the human ribosome. For cross-linking to ribosomal protein S15, a set of mRNA was used that contained triplet UUU/UUC at the 5'-termini and a perfluorophenyl azide-modified uridine in position 3' of this triplet. The locations of mRNA analogues on the ribosome were governed by tRNAPhe cognate to the UUU/UUC triplet targeted to the P site. Cross-linked S15 protein was isolated from the irradiated with mild UV light complexes of 80S ribosomes with tRNAPhe and mRNA analogues with subsequent cleavage with CNBr that splits polypeptide chain after methionines. Analysis of modified oligopeptides resulted from the cleavage revealed that in all cases cross-linking site was located in C-terminal fragment 111-145 of protein S15 indicating that this fragment is involved in formation of decoding site of the eukaryotic ribosome.     

Part of the matrix on the 5' side from codon in the E-segment is close to protein S26 on the human 80S ribosome

Positioning of mRNA on the 80S ribosome upstream the E site bound codon was studied using derivatives of nona- and dodecaribonucleotides containing the triplet UUU coding for Phe at the 3'-terminus, and a perfluorophenylazide cross-linker on either the first or the third nucleotide. Two sets of the mRNA analogues were used, with the photoactivatable groups on either the C5 atom of the uridine or the N7 atom of the guanosine. The modified nucleotides were directed to positions from -4 to -9 with respect to the first nucleotide of the P site bound codon by tRNA(Phe) cognate to the triplet UUU targeted to the P site. Mild UV-irradiation of ribosomecomplexes with tRNA(Phe) and mRNA analogues resulted in the cross-linking to the 40S subunits preferentially, mainly to the proteins. The principal target for the cross-linking was protein S26 in all cases. Location of the photoactivatable group on the nucleotide at position -4 lead also to the minor cross-linking to protein S3, and at position -6 to protein S14. In the absence of tRNA, all mRNA analogues cross-linked to protein S3.     

Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.     

Arrangement of the Sense and Stop Codons of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking with Oligonucleotide Derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3-flanking sense or stop codon with a perfluoroarylazido group at G or U were used to study the positioning of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, tRNA^Phe cognate to UCC was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 and nucleotide A1825 in helix 44 close to the 3 end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem–loop fragment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in the ribosomal A site of various organisms.     

The C domain of translation termination factor eRF1 is close to the stop codon in the A site of the 80S ribosome

The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome.     

Crosslinking of mRNA Analog,pUUAGUAUUUAUU Derivative with Photoactivatable Group at the Guanosine Residue,with Human 80S Ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg²⁺, or 4 mM Mg²⁺ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840–1849 of 18S rRNA, but in the complexes formed with participation of Phe-tRNA^Phe (where the G residue carrying the arylazido group occupied position –3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position –3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg²⁺ concentration and the presence of polyamines.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.