Studying functional significance of the sequence 980–1061 in the central domain of human 18S rRNA using complementary DNA probes

Graifer, D. M.; Malygin, Alexey A.; Matasova, N. B.; Mundus, D. A.; Zenkova, Marina A.; Карпова, Г. Г.

doi:10.1016/s0167-4781(96)00176-5

Cited by 32 publications

(10 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A pre-treatment of filters with 0.5 M KOH (25°C, 25 min) (21) proposed as solution for this trouble for short oligonucleotides did not work in our case for heteropolymeric mRNA (49 nt).…”

Section: Methodsmentioning

confidence: 67%

Features of 80S mammalian ribosome and its subunits

Budkevich

El'skaya

Nierhaus

2008

Nucleic Acids Research

View full text Add to dashboard Cite

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic.

show abstract

“…A pre-treatment of filters with 0.5 M KOH (25°C, 25 min) (21) proposed as solution for this trouble for short oligonucleotides did not work in our case for heteropolymeric mRNA (49 nt).…”

Section: Methodsmentioning

confidence: 67%

Features of 80S mammalian ribosome and its subunits

Budkevich

El'skaya

Nierhaus

2008

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…Binary complexes of HCV IRES and 40S subunits were obtained by incubating the subunits (2.0 × 10 −6 M if not specified otherwise) with IRES (0.5 × 10 −6 M if not specified otherwise) in buffer A at 37°C for 10 min. The extent of binding of the 5′- 32 P-labeled RNAs to 40S subunits was examined by the nitrocellulose filtration assay using alkali pretreated filters as described (31). In the experiments on the determination of cross-linked ribosomal proteins, the binary complexes were formed starting from 10–20 pmol of the derivatized HCV IRES.…”

Section: Methodsmentioning

confidence: 99%

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

Babaylova

Graifer

Malygin

et al. 2008

Nucleic Acids Research

View full text Add to dashboard Cite

The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

show abstract

“…Ribosomal complexes containing mRNAs, tRNAs, and release factors were obtained in buffer A at 20°C; the extent of mRNA binding was examined by nitrocellulose filtration with the use of the oligoribonucleotides labeled at the 5 ′ -termini by [γ-32 P]ATP and polynucleotide kinase (Graifer et al 1997). Binary complex 2 (lacking tRNA) was obtained by incubation of ribosomes (0.5 μM) with oligoribonucleotide UUC UAA AAG (5 μM) for 40 min.…”

Section: Formation and Analysis Of Ribosomal Complexesmentioning

confidence: 99%

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

Bulygin

Bartuli

Malygin

et al. 2015

RNA

Self Cite

View full text Add to dashboard Cite

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA-and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribosephosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.

show abstract

Studying functional significance of the sequence 980–1061 in the central domain of human 18S rRNA using complementary DNA probes

Cited by 32 publications

References 24 publications

Features of 80S mammalian ribosome and its subunits

Features of 80S mammalian ribosome and its subunits

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

Contact Info

Product

Resources

About