scholarly journals The Bulged Nucleotide in the Escherichia coli Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach

2000 ◽  
Vol 182 (22) ◽  
pp. 6302-6307 ◽  
Author(s):  
Chuang Li ◽  
Myriam Reches ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Insertion Sequence ◽  
Stop Codon ◽  
Loop Structure ◽  
Genetic Approach ◽  
Stem Loop ◽  
E Coli ◽  
Stem Loop Structure ◽  
Suppressor Mutations

ABSTRACT The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires acis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. InEscherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of thefdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizingselB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selBsuppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a “single suppressible unit,” suggesting that there is some flexibility to the nature of the interaction between this element and SelB.

scholarly journals Kinetics of Polynucleotide Phosphorylase: Comparison of Enzymes from Streptomyces and Escherichia coli and Effects of Nucleoside Diphosphates

2007 ◽  
Vol 190 (1) ◽  
pp. 98-106 ◽  
Author(s):  
Samantha A. Chang ◽  
Madeline Cozad ◽  
George A. Mackie ◽  
George H. Jones
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Coelicolor ◽  
Loop Structure ◽  
Polynucleotide Phosphorylase ◽  
Mobility Shift ◽  
Stem Loop ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
Stem Loop Structure ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The k cat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.

scholarly journals Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142 ◽  
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

A 3' Stem/Loop Structure of the Chlamydomonas Chloroplast atpB Gene Regulates mRNA Accumulation in vivo

1991 ◽  
Vol 3 (3) ◽  
pp. 285 ◽  
Author(s):  
David B. Stern ◽  
Elaine R. Radwanski ◽  
Karen L. Kindle
Keyword(s):  
Loop Structure ◽  
Mrna Accumulation ◽  
Stem Loop ◽  
Stem Loop Structure

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

scholarly journals Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

1999 ◽  
Vol 19 (1) ◽  
pp. 835-845 ◽  
Author(s):  
Zeng-Feng Wang ◽  
Thomas C. Ingledue ◽  
Zbigniew Dominski ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Translational Control ◽  
Mrna Processing ◽  
Loop Structure ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Stem Loop Structure ◽  
Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

scholarly journals The human box C/D snoRNA U3 is a miRNA source and miR-U3 regulates expression of sortin nexin 27

2020 ◽  
Vol 48 (14) ◽  
pp. 8074-8089
Author(s):  
Nicolas Lemus-Diaz ◽  
Rafael Rinaldi Ferreira ◽  
Katherine E Bohnsack ◽  
Jens Gruber ◽  
Markus T Bohnsack
Keyword(s):  
Mirna Biogenesis ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Fragments ◽  
Reporter Assays ◽  
Mirna Biogenesis Pathway

Abstract MicroRNAs (miRNAs) are important regulators of eukaryotic gene expression and their dysfunction is often associated with cancer. Alongside the canonical miRNA biogenesis pathway involving stepwise processing and export of pri- and pre-miRNA transcripts by the microprocessor complex, Exportin 5 and Dicer, several alternative mechanisms of miRNA production have been described. Here, we reveal that the atypical box C/D snoRNA U3, which functions as a scaffold during early ribosome assembly, is a miRNA source. We show that a unique stem–loop structure in the 5′ domain of U3 is processed to form short RNA fragments that associate with Argonaute. miR-U3 production is independent of Drosha, and an increased amount of U3 in the cytoplasm in the absence of Dicer suggests that a portion of the full length snoRNA is exported to the cytoplasm where it is efficiently processed into miRNAs. Using reporter assays, we demonstrate that miR-U3 can act as a low proficiency miRNA in vivo and our data support the 3′ UTR of the sortin nexin SNX27 mRNA as an endogenous U3-derived miRNA target. We further reveal that perturbation of U3 snoRNP assembly induces miR-U3 production, highlighting potential cross-regulation of target mRNA expression and ribosome production.

A Single Stem-Loop Structure within the HTLV-1 Rex Response Element Is Sufficient to Mediate Rex Activity in Vivo

Virology ◽  
1994 ◽  
Vol 204 (1) ◽  
pp. 144-152 ◽  
Author(s):  
Monika Gröne ◽  
Erich Hoffmann ◽  
Susanne Berchtold ◽  
Bryan R. Cullen ◽  
Ralph Grassmann
Keyword(s):  
Loop Structure ◽  
Response Element ◽  
Stem Loop ◽  
Stem Loop Structure

scholarly journals Importance of a 5′ Stem-Loop for Longevity ofpapA mRNA in Escherichia coli

1999 ◽  
Vol 181 (11) ◽  
pp. 3587-3590 ◽  
Author(s):  
Angela L. Bricker ◽  
Joel G. Belasco
Keyword(s):  
Escherichia Coli ◽  
Untranslated Region ◽  
High Level Expression ◽  
Large Measure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Significant Acceleration ◽  
Long Lifetime ◽  
High Level

ABSTRACT High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papAmessage begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5′ untranslated region, as evidenced by the significant acceleration inpapA mRNA decay that results from its removal.

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