Jin-Der Wen

National Taiwan University | NTU

Significance Ribosomes translate the genetic codes of messenger RNA (mRNA) to make proteins. Translation must begin at the correct initiation site; otherwise, abnormal proteins will be produced. Here, we show that a short ribosome-specific sequence in the upstream followed by an unstructured downstream sequence is a favorable initiation site. Those...

Programmed –1 ribosomal frameshifting is an essential regulation mechanism of translation in viruses and bacteria. It is stimulated by mRNA structures inside the coding region. As the structure is unfolded repeatedly by consecutive translating ribosomes, whether it can refold properly each time is important in performing its function. By using sing...

Programmed −1 ribosomal frameshifting (−1 PRF) is a translation mechanism that regulates the relative expression level of two proteins encoded on the same messenger RNA (mRNA). This regulation is commonly used by viruses such as coronaviruses and retroviruses but rarely by host human cells, and for this reason, it has long been considered as a ther...

Shine-Dalgarno (SD) sequences, the core element of prokaryotic ribosome-binding sites, facilitate mRNA translation by base-pair interaction with the anti-SD (aSD) sequence of 16S rRNA. In contrast to this paradigm, an inspection of thousands of prokaryotic species unravels tremendous SD sequence diversity both within and between genomes, whereas aS...

Shine-Dalgarno sequences (SD) in prokaryotic mRNA facilitate protein translation by pairing with rRNA in ribosomes. Although conventionally defined as AG-rich motifs, recent genomic surveys reveal great sequence diversity, questioning how SD functions. Here, we determined the molecular fitness (i.e., translation efficiency) of 49 synthetic 9-nt SD...

Motivation: Programmed ribosomal frameshifting (PRF) is widely used by viruses and bacteria to produce different proteins from a single mRNA template. How steric hindrance of a PRF-stimulatory mRNA structure transiently modifies the conformational dynamics of the ribosome, and thereby allows tRNA slippage, remains elusive. Results: Here, we leve...

Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating...

Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, wh...

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within...

Folding messenger RNA into specific structures is a common regulatory mechanism involved in translation. In Escherichia coli, the operator of the rpsO gene transcript folds into a pseudoknot or double-hairpin conformation. S15, the gene product, binds only to the pseudoknot, thereby repressing its own synthesis when it is present in excess in the c...

Translation initiation of mRNA can be regulated through different ways in the cell. One of the common mechanisms is to modulate the structural elements of mRNA. Escherichia coli ribosomal protein S15 (ecS15, encoded by the rpsO gene) regulates its own biosynthesis by interacting with the 5' untranslated region (5'-UTR) of its cognate mRNA. When ecS...

Translocation of tRNA and mRNA by the ribosome during protein synthesis involves a number of precise and coordinated macromolecular rearrangements. Recently, high-resolution x-ray structures, cryo-EM reconstructions, and single-molecule fluorescence studies have yielded insight into the nature of these conformational states, and the kinetics of the...

Secondary and tertiary structures of mRNA may become barriers to the ribosome during translation. Previous studies have shown that the ribosome itself is capable of opening the base-paring of mRNA. More recently, by using optical tweezers, Qu et al. have shown that this unwinding process involves two kinds of active mechanisms, in which the ribosom...

The dnaX gene in Escherichia coli encodes comparable amounts of τ and γ subunits of DNA polymerase III by programmed ribosomal frameshifting (PRF). An early termination codon in the −1 frame of dnaX produces the γ subunit, which is a truncated form of the τ subunit. PRF is also involved in protein expression in many viruses, such as the Gag-Pol pol...

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures ar...

Ribosomes translate the genetic code in an mRNA into a protein; three nucleotides-one codon-code for one amino acid. Because natural mRNAs contain base-paired regions, the ribosome needs to unwind these structures into single-strands before the structured regions can be translated. Furthermore, mRNA secondary and tertiary structures are involved in...

Programmed ribosomal frameshifting is involved in regulation of gene expression at the translation level in bacteria, and the frameshifting efficiency has to be well controlled. For example, the dnaX gene of E. coli encodes two subunits (gamma and tau) of the DNA polymerase III, and the ratio of these two subunits is determined by the frameshifting...

In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed...

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each paus...

The synthesis of proteins, translation, is carried out by ribosomes. Translation is a dynamic process whereby codons in the messenger RNA (mRNA) are "read" one at a time by base pairing with transfer RNA (tRNA) molecules and the corresponding amino acids are polymerized sequentially forming polypeptides. To date, the dynamics of translation, in par...

Circular dichroism (CD) spectroscopy is widely used to characterize the structures of DNA G-quadruplexes. CD bands at 200-300 nm have been empirically related to G-quadruplexes having parallel or antiparallel sugar-phosphate backbones. We propose that a more fundamental interpretation of the origin of the CD bands is in the stacking interactions of...

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state f...

By exerting mechanical force it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model incl...

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model inc...

Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed whil...

The gene 5 protein (g5p) encoded by filamentous Ff phages is an ssDNA-binding protein, which binds to and sequesters the nascent ssDNA phage genome in the process of phage morphogenesis. The g5p also binds with high affinity to DNA and RNA sequences that form G-quadruplex structures. However, sequences that would form G-quadruplexes are absent in s...

Single-stranded DNA or RNA libraries used in SELEX experiments usually include primer-annealing sequences for PCR amplification. In genomic SELEX, these fixed sequences may form base pairs with the central genomic fragments and interfere with the binding of target molecules to the genomic sequences. In this study, a method has been developed to cir...

The Ff gene 5 protein (g5p) is classified as a single-stranded DNA-binding protein. However, we previously showed that g5p binds with high affinity to a SELEX-selected G-rich 58-mer DNA oligomer, I-3, that forms an intramolecular G-quadruplex [Wen, J.-D., Gray, C. W., and Gray, D. M. (2001) Biochemistry 40, 9300-9310]. In 200 mM NaCl at 37 degrees...

We review CD studies of a single-stranded DNA binding protein, g5p, of the Ff group of bacterial viruses. The CD spectrum of the g5p is dominated by a positive tyrosine La band at 229 nm, to which all five of the protein tyrosines contribute. The La band becomes much less positive upon binding of g5p to nucleic acids. CD spectra of mutant proteins...

We review CD studies of a single-stranded DNA binding protein, gyp, of the Ff group of bacterial viruses. The CD spectrum of the gyp is dominated by a positive tyrosine L-a band at 229 rim, to which all five of the protein tyrosines contribute. The L-a band becomes much less positive upon binding of gyp to nucleic acids. CD spectra of mutant protei...

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight round...

Several investigations have shown that the renal medulla has a greater capacity to generate nitric oxide than the renal cortex. To further evaluate the changes of nitric oxide synthesis in the kidney, particularly in the outer medulla, in disorders involving fluid and electrolyte imbalances, we sought to determine renal nitric oxide synthase expres...

Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats. Experiments were performed to investigate whether renal nitric oxide synthase (NOS) mRNA and protein expression are responsive to the alteration of body volume. Four days of water deprivation (WD) was initiated in 16 male Wistar rats, and 16 normal rats (NC...

The pyr*pur·pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crickpaired with an antiparallel pyrimidine strand (pur·pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paire...

Experiments were performed to examine the effect of water deprivation and salt restriction on ANP synthesis in the kidneys and hearts of normal rats. A 4-day water deprivation (WD) and 7-day salt restriction (SR; 0.01% NaCl) were performed in 12 and 14 rats, respectively. Atrial natriuretic peptide (ANP) mRNA expression in the kidney was assessed w...

To investigate the responsiveness of renal-synthesized C-type natriuretic peptide (CNP) to changes in water and electrolyte balance, we measured renal CNP mRNA levels, plasma CNP concentrations and urinary CNP excretion rates in streptozotocin-induced diabetic rats eating a normal (0.26% NaCl) or low (0.04% NaCl) salt diet. Using reverse transcript...