A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

2004 ◽  
Vol 36 (4) ◽  
pp. 259-268 ◽  
Author(s):  
Li Yang ◽  
Jiang Chen ◽  
Catherine C. Y. Chang ◽  
Xin-Ying Yang ◽  
Zhen-Zhen Wang ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Loop Structure ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Codon Selection ◽  
Mutation Analyses ◽  
Enzymatic Activity Assay ◽  
Selection Of

Abstract Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397–1399 and AUG1415–1417, at 5′-terminus of the open reading frame (ORF, nt 1397–3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397–1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397–1399 as a main start codon, in addition to upstream nucleotide A in the –3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.

scholarly journals Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

scholarly journals Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

2009 ◽  
Vol 84 (1) ◽  
pp. 52-58 ◽  
Author(s):  
Seahee Kim ◽  
Haifeng Wang ◽  
Wang-Shick Ryu
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Loop Structure ◽  
Stem Loop ◽  
Eukaryotic Translation ◽  
Stem Loop Structure ◽  
B Virus ◽  
Rnp Complex ◽  
Eukaryotic Translation Initiation

ABSTRACT The DNA genome of hepatitis B virus (HBV) replicates via reverse transcription within capsids following the encapsidation of an RNA template, the pregenomic RNA (pgRNA). We previously demonstrated that the 5′ cap proximity of the stem-loop structure (ε or epsilon), an encapsidation signal, is critically important for the encapsidation of the pgRNA (J. K. Jeong, G. S. Yoon, and W. S. Ryu, J. Virol. 74:5502-5508, 2000). Therefore, we speculated that the viral polymerase (Pol), while bound to the 5′ ε stem-loop structure, could recognize the cap via its interaction with eIF4E, a eukaryotic translation initiation factor. Our data showed the direct interaction between HBV Pol and eIF4E, as measured by coimmunoprecipitation. Further, we demonstrated that eIF4E interacts with the Pol-ε ribonucleoprotein complex (RNP) rather than Pol alone, resulting in eIF4E-Pol-ε RNP complex formation. In addition, we asked whether eIF4E remains engaged to the Pol-ε RNP complex during nucleocapsid assembly. Density gradient analysis revealed that eIF4E indeed was incorporated into nucleocapsids. It is of great importance to uncover whether the incorporated eIF4E contributes to viral reverse transcription or other steps in the HBV life cycle.

scholarly journals A Translation-Aborting Small Open Reading Frame in the Intergenic Region Promotes Translation of a Mg2+ Transporter in Salmonella Typhimurium

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Eunna Choi ◽  
Yoontak Han ◽  
Shinae Park ◽  
Hyojeong Koo ◽  
Jung-Shin Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Translation Initiation ◽  
Intergenic Region ◽  
Ribosomal Subunit ◽  
Open Reading Frame ◽  
Start Codon ◽  
Stem Loop ◽  
Reading Frame ◽  
Ribosome Binding

ABSTRACT Bacterial mRNAs often harbor upstream open reading frames (uORFs) in the 5′ untranslated regions (UTRs). Translation of the uORF usually affects downstream gene expression at the levels of transcription and/or translation initiation. Unlike other uORFs mostly located in the 5′ UTR, we discovered an 8-amino-acid ORF, designated mgtQ, in the intergenic region between the mgtC virulence gene and the mgtB Mg2+ transporter gene in the Salmonella mgtCBRU operon. Translation of mgtQ promotes downstream mgtB Mg2+ transporter expression at the level of translation by releasing the ribosome-binding sequence of the mgtB gene that is sequestered in a translation-inhibitory stem-loop structure. Interestingly, mgtQ Asp2 and Glu5 codons that induce ribosome destabilization are required for mgtQ-mediated mgtB translation. Moreover, the mgtQ Asp and Glu codons-mediated mgtB translation is counteracted by the ribosomal subunit L31 that stabilizes ribosome. Substitution of the Asp2 and Glu5 codons in mgtQ decreases MgtB Mg2+ transporter production and thus attenuates Salmonella virulence in mice, likely by limiting Mg2+ acquisition during infection. IMPORTANCE Translation initiation regions in mRNAs that include the ribosome-binding site (RBS) and the start codon are often sequestered within a secondary structure. Therefore, to initiate protein synthesis, the mRNA secondary structure must be unfolded to allow the RBS to be accessible to the ribosome. Such unfolding can be achieved by various mechanisms that include translation of a small upstream open reading frame (uORF). In the intracellular pathogen Salmonella enterica serovar Typhimurium, translation of the Mg2+ transporter mgtB gene is enhanced by an 8-amino-acid upstream ORF, namely, mgtQ, that harbors Asp and Glu codons, which are likely to destabilize ribosome during translation. Translation of the mgtQ ORF promotes the formation of a stem-loop mRNA structure sequestering anti-RBS and thus releases the mgtB RBS. Because mgtQ-mediated MgtB Mg2+ transporter production is required for Salmonella virulence, this pathogen seems to control the virulence determinant production exquisitely via this uORF during infection.

scholarly journals Bi-directional ribosome scanning controls the stringency of start codon selection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yifei Gu ◽  
Yuanhui Mao ◽  
Longfei Jia ◽  
Leiming Dong ◽  
Shu-Bing Qian
Keyword(s):  
Translation Initiation ◽  
Current Knowledge ◽  
Start Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Operational Mechanism ◽  
Codon Recognition ◽  
Codon Selection ◽  
Selection Of

AbstractThe fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5ʹ→3ʹ migration of the pre-initiation complex (PIC) along the 5ʹ UTR. In probing translation initiation from ultra-short 5ʹ UTR, we report that an AUG triplet near the 5ʹ end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5ʹ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5ʹ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.

scholarly journals A Group IIC-Type Intron Interrupts the rRNA Methylase Gene of Geobacillus stearothermophilus Strain 10

2010 ◽  
Vol 192 (19) ◽  
pp. 5245-5248 ◽  
Author(s):  
Samuel E. Moretz ◽  
Bert C. Lampson
Keyword(s):  
Open Reading Frame ◽  
Loop Structure ◽  
Geobacillus Stearothermophilus ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Insertion Sites ◽  
Methylase Gene

ABSTRACT Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from Geobacillus stearothermophilus were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.

scholarly journals Complete Genome Sequence of Beak and Feather Disease Virus Isolated from an African Grey Parrot in China in 2017

2018 ◽  
Vol 6 (6) ◽  
pp. e01519-17
Author(s):  
Xuejun Guo ◽  
Xinna Ge ◽  
Jianyu Chang
Keyword(s):  
Disease Virus ◽  
Viral Genome ◽  
Loop Structure ◽  
Stem Loop ◽  
Reading Frame ◽  
African Grey Parrot ◽  
Stem Loop Structure ◽  
Grey Parrot ◽  
Feather Disease Virus ◽  
Open Reading Frame 1

ABSTRACT The complete nucleotide (nt) sequence of beak and feather disease virus (BFDV) was determined. The viral genome consists of 1,991 nt, including an 870-nt open reading frame 1 (ORF1), a 744-nt ORF2, a conserved stem-loop structure, and the second hairpin. This is the first reported detection of BFDV in an infected African grey parrot in China.

scholarly journals Translation of the Flagellar Gene fliO ofSalmonella typhimurium from Putative Tandem Starts

1998 ◽  
Vol 180 (11) ◽  
pp. 2936-2942 ◽  
Author(s):  
Gary J. Schoenhals ◽  
May Kihara ◽  
Robert M. Macnab
Keyword(s):  
Start Codon ◽  
Loop Structure ◽  
Flagellar Gene ◽  
Transmembrane Segment ◽  
Stem Loop ◽  
E Coli ◽  
Protein Levels ◽  
Stem Loop Structure ◽  
Loop Sequence ◽  
Entire Sequence

ABSTRACT The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092–6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189–197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis genecheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found withcheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

scholarly journals Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

1999 ◽  
Vol 19 (6) ◽  
pp. 4334-4342 ◽  
Author(s):  
B. Stefanovic ◽  
C. Hellerbrand ◽  
D. A. Brenner
Keyword(s):  
Protein Complexes ◽  
Stellate Cell ◽  
State Level ◽  
Start Codon ◽  
Loop Structure ◽  
Fibrillar Collagen ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reporter Mrna

ABSTRACT Three fibrillar collagen mRNAs, α1(I), α2(I), and α1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen α1(I) reporter mRNAs in hsc’s and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc’s and to a lesser extent in activated hsc’s and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc’s and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3′ untranslated region (UTR) of collagen α1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc’s. This 3′ UTR binds αCP, a protein implicated in increasing stability of collagen α1(I) mRNA in activated hsc’s (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201–5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen α1(I) mRNA is regulated by a complex interaction between the 5′ stem-loop and the 3′ UTR, which may optimize collagen production in activated hsc’s.

Sign in / Sign up

Export Citation Format

Share Document