Catalytic roles of the AMP at the 3′ end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3 terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3 terminus of tRNA.     

Effect of removal of 160 nucleotides from the 3′ end of Escherichia coli 16S rRNA on the reconstitution and activity of 30S ribosomes

$\text{E. coli}$ 16S rRNA deprived of 160 nucleotides from its 3′ end was obtained by digestion with polynucleotide phosphorylase. Such rRNA was used for the reconstitution of 30S subunits and the resulting particles contained all proteins present in native 30S ribosomes. Their sedimentation coefficient was estimated as 26.5S. Poly AUG-dependent binding of fMet-tRNA to subunits reconstituted with shortened rRNA was the same as to 30S particles reconstituted with the native 16S rRNA. Subunits reconstituted with shortened rRNA were also active in poly U-dependent phenylalanine incorporation; however, their activity reached only 50% of that obtained with 30S subunits reconstituted with native 16S rRNA.     

The identification of cDNA clones that include the 3′ end of mRNA

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3′ end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.     

Nucleotide sequence of two 3′-termini of rRNA in the sea urchin Lytechinus variegatus

The 3′-terminus of 26 S rRNA from the sea urchin Lytechinus variegatus has been determined by oligonucleotide fingerprinting, S1 nuclease mapping and terminal nucleotide analysis. There are two species of 26 S rRNA of approximately equal abundance, one 19 nucleotides longer than the other.     

Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3′ terminal cloacin fragment of 16 S rRNA from nuclease S1

Footprinting of ribosomal protein S1 on the 49-nucleotide 3′ terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1.     

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.     

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.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

An informative polymorphism detectable by polymerase chain reaction at the 3′ end of the dystrophin gene

Summary A fragment that contains a (CA)n sequence from the 3 untranslated region of the dystrophin gene can be amplified by the polymerase chain reaction and shows length polymorphism in a Caucasian population. The two common alleles differ by 4bp. This new genetic marker has a heterozygosity of about 35% and is typed more rapidly than a conventional restriction fragment length polymorphism. Its localisation at the 3 end of the dystrophin gene makes it a useful tool for diagnostic applications in families with Duchenne/ Becker muscular dystrophy, and for the analysis of intragenic recombination.     

Identification of recombinant plasmids containing DNA sequences derived from the 3′ end of ovine thyroglobulin mRNA

This paper describes the cloning inE. coli of several DNA copies of the 3 portion of ovine thyroglobulin mRNA. The cDNA clones were identified byin situ cloning hybridization to³²P-labelled thyroglobulin cDNA and by positive hybridization-translation assays. The longest thyroglobulin cDNA fragment cloned (psTg21A, 1670 bp), was shown by S₁ nuclease mapping to be an unaltered copy of the thyroglobulin mRNA. psTg 21A overlapped with another cDNA fragment (psTg 11, 330 bp) containing the poly(dA)-tail and corresponding therefore to the 3 end of the mRNA. When aligned together the two clones represent more than 20% of the thyroglobulin mRNA length. Another cDNA fragment (psTg 15, 350 bp) was identified as an internal portion of the thyroglobulin mRNA sequence.Abbreviations Amp^sTe^r = ampicillin sensitive, tetracycline resistant - bp = base pairs - EDTA = ethylene diamine tetra acetic acid - Pipes = 1,4-piperazine diethane sulfonic acid - SDS = sodium dodecylsulfate