data_5980 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Structure Function Analysis of the Stemp Loop IIIc of HCV and GBV-B IRES ; _BMRB_accession_number 5980 _BMRB_flat_file_name bmr5980.str _Entry_type original _Submission_date 2003-10-22 _Accession_date 2003-10-22 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details . loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Thiviyanathan Varatharasa . . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 71 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2004-11-08 original author . stop_ _Original_release_date 2004-11-08 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites. ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 15476802 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Rijnbrand R. . . 2 Thiviyanathan Varatharasa . . 3 Kaluarachchi K. . . 4 Lemon S. M. . 5 Gorenstein D. G. . stop_ _Journal_abbreviation 'J. Mol. Biol.' _Journal_volume 343 _Journal_issue 4 _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first 805 _Page_last 817 _Year 2004 _Details . loop_ _Keyword 'GB Virus' IRES 'stem loop' stop_ save_ ####################################### # Cited references within the entry # ####################################### save_ref_1 _Saveframe_category citation _Citation_full ; Honda M, Beard MR, Ping LH, Lemon SM. J. Virol., (1999) 73, 1165-1174. ; _Citation_title "A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation." _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9882318 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Honda M . . 2 Beard 'M R' R. . 3 Ping 'L H' H. . 4 Lemon 'S M' M. . stop_ _Journal_abbreviation 'J. Virol.' _Journal_name_full 'Journal of virology' _Journal_volume 73 _Journal_issue 2 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 1165 _Page_last 1174 _Year 1999 _Details ; Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5' nontranslated RNA (5'NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5' border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mutational analysis of putative helical structures that examined the impact of both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to 52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV 5'NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cleavage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly dependent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserved helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests that these segments of the viral RNA may interact with elements of the host translational machinery that are broadly conserved among different mammalian species. ; save_ ################################## # Molecular system description # ################################## save_IRES _Saveframe_category molecular_system _Mol_system_name IRES _Abbreviation_common IRES _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label 'IIIc, monomer' $RNA stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state monomer _System_paramagnetic no _System_thiol_state 'not present' _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_RNA _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class RNA _Name_common 'Ribo Nucleic Acid' _Abbreviation_common RNA _Molecular_mass . _Mol_thiol_state 'not present' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 10 _Mol_residue_sequence GGGCAAGCCC loop_ _Residue_seq_code _Residue_label 1 G 2 G 3 G 4 C 5 A 6 A 7 G 8 C 9 C 10 C stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $RNA . . viruses . 'GB Virus' B stop_ save_ ######################### # Experimental source # ######################### save_experimental_source _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_name $RNA 'enzymatic semisynthesis' . . . . . stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Concentration_min_value _Concentration_max_value _Isotopic_labeling $RNA . mM 0.2 0.8 . stop_ save_ ############################ # Computer software used # ############################ save_FELIX _Saveframe_category software _Name FELIX _Version 97 loop_ _Task 'peak assignemnt' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model UnityPlus _Field_strength 600 _Details . save_ save_NMR_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Varian _Model UnityPlus _Field_strength 750 _Details . save_ ############################# # NMR applied experiments # ############################# save_2D_NOESY,_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D NOESY,' _Sample_label . save_ save_2D_TOCSY_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D TOCSY' _Sample_label . save_ save_1H-31P_HETRO_TOCSY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '1H-31P HETRO TOCSY' _Sample_label . save_ save_DQF-COSY_4 _Saveframe_category NMR_applied_experiment _Experiment_name DQF-COSY _Sample_label . save_ save_NMR_spec_expt__0_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D NOESY,' _BMRB_pulse_sequence_accession_number . _Details . save_ save_NMR_spec_expt__0_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D TOCSY' _BMRB_pulse_sequence_accession_number . _Details . save_ save_NMR_spec_expt__0_3 _Saveframe_category NMR_applied_experiment _Experiment_name '1H-31P HETRO TOCSY' _BMRB_pulse_sequence_accession_number . _Details . save_ save_NMR_spec_expt__0_4 _Saveframe_category NMR_applied_experiment _Experiment_name DQF-COSY _BMRB_pulse_sequence_accession_number . _Details . save_ ####################### # Sample conditions # ####################### save_conidtion_1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units pH 6.8 0.05 n/a temperature 298 1 K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chemical_shift_reference _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio water H 1 protons ppm 4.76 internal direct spherical internal parallel 1 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_shift_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $conidtion_1 _Chem_shift_reference_set_label $chemical_shift_reference _Mol_system_component_name 'IIIc, monomer' _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 . 1 G H1 H 12.88 0.01 1 2 . 1 G H8 H 8.19 0.01 1 3 . 1 G H1' H 5.90 0.01 1 4 . 1 G H2' H 4.98 0.01 1 5 . 1 G H3' H 4.62 0.01 1 6 . 1 G H4' H 4.54 0.01 1 7 . 2 G H1 H 12.83 0.01 1 8 . 2 G H8 H 7.62 0.01 1 9 . 2 G H1' H 6.00 0.01 1 10 . 2 G H2' H 4.62 0.01 1 11 . 2 G H3' H 4.53 0.01 1 12 . 2 G H4' H 4.49 0.01 1 13 . 3 G H1 H 13.24 0.01 1 14 . 3 G H8 H 7.29 0.01 1 15 . 3 G H1' H 5.84 0.01 1 16 . 3 G H2' H 4.59 0.01 1 17 . 3 G H3' H 4.54 0.01 1 18 . 3 G H4' H 4.45 0.01 1 19 . 3 G H5' H 4.21 0.01 2 20 . 3 G H5'' H 4.20 0.01 2 21 . 4 C H41 H 8.05 0.01 2 22 . 4 C H42 H 6.79 0.01 2 23 . 4 C H5 H 5.12 0.01 1 24 . 4 C H6 H 7.41 0.01 1 25 . 4 C H1' H 5.52 0.01 1 26 . 4 C H2' H 4.66 0.01 1 27 . 4 C H3' H 4.44 0.01 1 28 . 4 C H4' H 4.28 0.01 1 29 . 5 A H2 H 8.13 0.01 1 30 . 5 A H8 H 8.19 0.01 1 31 . 5 A H1' H 6.01 0.01 1 32 . 5 A H2' H 4.62 0.01 1 33 . 5 A H3' H 4.27 0.01 1 34 . 5 A H4' H 4.21 0.01 1 35 . 6 A H2 H 7.93 0.01 1 36 . 6 A H8 H 7.86 0.01 1 37 . 6 A H1' H 5.94 0.01 1 38 . 6 A H2' H 4.87 0.01 1 39 . 6 A H3' H 4.66 0.01 1 40 . 6 A H4' H 4.51 0.01 1 41 . 6 A H5' H 4.31 0.01 2 42 . 7 G H1 H 12.84 0.01 1 43 . 7 G H8 H 7.50 0.01 1 44 . 7 G H1' H 5.22 0.01 1 45 . 7 G H2' H 4.42 0.01 1 46 . 7 G H3' H 4.37 0.01 1 47 . 7 G H4' H 4.17 0.01 1 48 . 8 C H41 H 8.61 0.01 2 49 . 8 C H42 H 6.92 0.01 2 50 . 8 C H5 H 5.22 0.01 1 51 . 8 C H6 H 7.67 0.01 1 52 . 8 C H1' H 5.50 0.01 1 53 . 8 C H2' H 4.46 0.01 1 54 . 8 C H3' H 4.32 0.01 1 55 . 8 C H4' H 4.28 0.01 1 56 . 9 C H41 H 8.58 0.01 2 57 . 9 C H42 H 6.98 0.01 2 58 . 9 C H5 H 5.51 0.01 1 59 . 9 C H6 H 7.84 0.01 1 60 . 9 C H1' H 5.55 0.01 1 61 . 9 C H2' H 4.55 0.01 1 62 . 9 C H3' H 4.32 0.01 1 63 . 9 C H4' H 4.22 0.01 1 64 . 10 C H41 H 8.32 0.01 2 65 . 10 C H42 H 7.13 0.01 2 66 . 10 C H5 H 5.56 0.01 1 67 . 10 C H6 H 7.74 0.01 1 68 . 10 C H1' H 5.81 0.01 1 69 . 10 C H2' H 4.06 0.01 1 70 . 10 C H3' H 4.23 0.01 1 71 . 10 C H4' H 4.32 0.01 1 stop_ save_