Peptide Elongation Rate Research Articles

A stochastic model for simulating ribosome kinetics in vivo.

Computational modelling of in vivo protein synthesis is highly complicated, as it requires the simulation of ribosomal movement over the entire transcriptome, as well as consideration of the concentration effects from 40+ different types of tRNAs and numerous other protein factors. Here I report on the development of a stochastic model for protein translation that is capable of simulating the dynamical process of in vivo protein synthesis in a prokaryotic cell containing several thousand unique mRNA sequences, with explicit nucleotide information for each, and report on a number of biological predictions which are beyond the scope of existing models. In particular, I show that, when the complex network of concentration dependent interactions between elongation factors, tRNAs, ribosomes, and other factors required for protein synthesis are included in full detail, several biological phenomena, such as the increasing peptide elongation rate with bacterial growth rate, are predicted as emergent properties of the model. The stochastic model presented here demonstrates the importance of considering the translational process at this level of detail, and provides a platform to interrogate various aspects of translation that are difficult to study in more coarse-grained models.

Single Molecule Measurement of Peptide Elongation Rate during Synthesis of a Full-Length Protein

Single Molecule Measurement of Peptide Elongation Rate during Synthesis of a Full-Length Protein

Single Molecule Measurement of Peptide Elongation Rate During Synthesis of a Full-Length Protein

The rate of translation of full-length proteins by the ribosome influences expression levels, folding and frameshifting. In order to study the factors that control translation rates, we use the expression of fast maturing Emerald Green Fluorescent Protein (EmGFP) by a reconsitituted E. coli cell-free translation system. Single-molecule TIRF microscopy allows monitoring of the appearance of single EmGFPs on the slide surface. Active translational complexes are docked to the microscope slide by a surface-immobilized antibody against an N-terminal extension of the translated protein. The appearance of fluorescent EmGFP spots identifies the end-point of full-length expression. In order to follow the translation rate in real time, the existing ribosomes and Phe-tRNAPhe in the cell-free mixture are replaced by fluorescent labeled Phe-tRNAPhe(Cy5.5) and L11(Cy3)-ribosomes, both of which are active in ensemble EmGFP expression assays. Individual Phe-tRNAPhes accommodated into the ribosome A-site during EmGFP translation are detected by single-molecule FRET pulses from Phe-tRNAPhe(Cy5.5) binding near the ribosomal protein L11(Cy3). Multiple accommodations of Phe-tRNAPhes on single ribosomes are observed during synthesis of EmGFP. The time intervals between two consecutive Phe-tRNAPhe accommodations can be assigned to particular sequence segments according to their temporal positions relative to two characteristic Phe-Phe doublets near the center of the EmGFP sequence. The assignments are validated by a decrease in translation rate when natural codons are replaced with codons pairing with rare isoacceptor tRNAs. This experimental platform will enable further testing of the control of translation rate by other rare codons, mRNA secondary structures, nascent polypeptide interactions with the ribosomal exit tunnel, and internal Shine-Dalgarno sequences. Supported by NIH Grant GM080376 and HFSP.

The ribosome uses two active mechanisms to unwind messenger RNA during translation

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 slows the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to enable translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting1,2, protein expression levels3,4, ribosome localization5, and cotranslational protein folding6. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases7, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding center is greatly influenced by the G•C content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favor its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: (i) it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations toward the open state, increasing the probability for the ribosome to translocate unhindered; and (ii) it also mechanically pulls apart the mRNA single-strands of the closed junction during the conformational changes that accompany ribosome translocation. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNAfMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.

Ribosomal Analysis of Rapid Rates of Protein Synthesis in the Antarctic Sea UrchinSterechinus neumayeri

Previous research has shown that developing stages of the Antarctic sea urchin Sterechinus neumayeri have high rates of protein synthesis that are comparable to those of similar species living in much warmer waters. Direct measurements of the biosynthetic capacities of isolated ribosomes have not been reported for marine organisms living in the extreme-cold environment of Antarctica. Such measurements are required for a mechanistic understanding of how the critical and highly complex processes involved in protein synthesis are regulated in animals living in the coldest marine environment on Earth (< -1 degrees C). We tested the hypothesis that high rates of protein synthesis in the cold are a direct result of high biosynthetic capacities of ribosomes engaged in protein synthesis. Our results show that the rate at which ribosomes manufacture proteins (i.e., the peptide elongation rate) at -1 degrees C is surprisingly similar to rates measured in other sea urchin species at temperatures that are over 15 degrees C warmer. Average peptide elongation rates for a range of developmental stages of the Antarctic sea urchin were 0.36 codons s(-1) (+/- 0.05, SE). On the basis of subcellular rate determinations of ribosomal activity, we calculated stage-specific rates of protein synthesis for blastulae and gastrulae to be 3.7 and 6.5 ng protein h(-1), respectively. These findings support the conclusion that the high rates of biosynthesis previously reported for the Antarctic sea urchin are an outcome of high ribosomal activities.

RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis.

BackgroundMean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different conditions of exponential growth.ResultsIt was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes). Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms.ConclusionOur results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and increased transcription of cellular maintenance genes at the expense of growth related genes. The strong link between P:N stoichiometry, RNA:protein ratio, ribosomal requirement for protein synthesis, and growth rate of microorganisms indicated by the study could be used to characterize the N and P economy of complex ecosystems such as soils and the oceans.

Characterizing L4 and L22 mutations in Escherichia coli strains resistant to Oleandomycin Triacetate

Ribosomes, essential to the life of the cell, are the site of protein synthesis. The bacterial ribosome contains two dissimilar subunits, 30S and 50S, that consist of rRNA molecules and proteins (ribosomal proteins). During translation, the ribosome mediates precise binding of the mRNA codon to the anticodon of a tRNA molecule containing a specific amino acid, and then catalyzes addition of the amino acid to the growing peptide chain. Macrolide antibiotics, such as erythromycin, inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit in the peptide exit channel. Interestingly, many ribosomal proteins such as L4 and L22 have extensions (tentacles) that reach into the mass of rRNA. The ends of the tentacles of L4 and L22 form a constriction in the peptide exit tunnel. Mutations in the L4 and L22 tentacles result in erythromycin resistance in E.coli, either because the antibiotic cannot bind (L4 mutant) or because it binds but cannot inhibit protein synthesis (L22 mutant). To learn more about the role of the tentacles in ribosome function and synthesis, we are isolating mutants of E. coli that are resistant to oleandomycin triacetate, a macrolide related to erythromycin. Mutants shown to have a change in the L4 or L22 gene will be characterized further. For example, assays will determine temperature sensitivity, growth rate, and rate of peptide elongation. The effect of the mutations on ribosome assembly and accuracy of translation will also be analyzed. (This research is supported by NSF Grant MCB-0349443 and MARC U*STAR Grant GM 08663 from NIH.)

The activity of ribosome modulation factor during growth of Escherichia coli under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

Ribosome Stalling Regulates IRES-Mediated Translation in Eukaryotes, a Parallel to Prokaryotic Attenuation

It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and remodeling of the leader to form an active IRES (the “zipper model” of translational control). It is shown here that slowing of the leader peptide elongation rate, either by cycloheximide or the introduction of rare codons, stimulated translation of the downstream ORF. These results suggest that ribosome stalling in the upstream ORF causes mRNA remodeling and formation of an active IRES. This control is reminiscent of translation attenuation in prokaryotic operons, where inhibition of translation elongation can regulate both mRNA translation and gene transcription by altering mRNA structure.

Mutation and selection on the anticodon of tRNA genes in vertebrate mitochondrial genomes

The H-strand of vertebrate mitochondrial DNA is left single-stranded for hours during the slow DNA replication. This facilitates C→U mutations on the H-strand (and consequently G→A mutations on the L-strand) via spontaneous deamination which occurs much more frequently on single-stranded than on double-stranded DNA. For the 12 coding sequences (CDS) collinear with the L-strand, NNY synonymous codon families (where N stands for any of the four nucleotides and Y stands for either C or U) end mostly with C, and NNR and NNN codon families (where R stands for either A or G) end mostly with A. For the lone ND6 gene on the other strand, the codon bias is the opposite, with NNY codon families ending mostly with U and NNR and NNN codon families ending mostly with G. These patterns are consistent with the strand-specific mutation bias. The codon usage biased towards C-ending and A-ending in the 12 CDS sequences affects the codon–anticodon adaptation. The wobble site of the anticodon is always G for NNY codon families dominated by C-ending codons and U for NNR and NNN codon families dominated by A-ending codons. The only, but consistent, exception is the anticodon of tRNA-Met which consistently has a 5′-CAU-3′ anticodon base-pairing with the AUG codon (the translation initiation codon) instead of the more frequent AUA. The observed CAU anticodon (matching AUG) would increase the rate of translation initiation but would reduce the rate of peptide elongation because most methionine codons are AUA, whereas the unobserved UAU anticodon (matching AUA) would increase the elongation rate at the cost of translation initiation rate. The consistent CAU anticodon in tRNA-Met suggests the importance of maximizing the rate of translation initiation.

Effect of temperature on in vivo protein synthetic capacity in Escherichia coli.

In this report, we examine the effect of temperature on protein synthesis. The rate of protein accumulation is determined by three factors: the number of working ribosomes, the rate at which ribosomes are working, and the rate of protein degradation. Measurements of RNA/protein ratios and the levels of individual ribosomal proteins and rRNA show that the cellular amount of ribosomal machinery in Escherichia coli is constant between 25 and 37 degreesC. Within this range, in a given medium, temperature affects ribosomal function the same as it affects overall growth. Two independent methodologies show that the peptide chain elongation rate increases as a function of temperature identically to growth rate up to 37 degreesC. Unlike the growth rate, however, the elongation rate continues to increase up to 44 degreesC at the same rate as between 25 and 37 degreesC. Our results show that the peptide elongation rate is not rate limiting for growth at high temperature. Taking into consideration the number of ribosomes per unit of cell mass, there is an apparent excess of protein synthetic capacity in these cells, indicating a dramatic increase in protein degradation at high temperature. Temperature shift experiments show that peptide chain elongation rate increases immediately, which supports a mechanism of heat shock response induction in which an increase in unfolded, newly translated protein induces this response. In addition, we find that at low temperature (15 degreesC), cells contain a pool of nontranslating ribosomes which do not contribute to cell growth, supporting the idea that there is a defect in initiation at low temperature.

Determination of the peptide elongation rate in vivo.

Determination of the peptide elongation rate in vivo.

The beneficial effects of incubating bovine embryos in groups are due to platelet-derived growth factor

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗dt = M·f·n·At, where dP∗dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and At = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis (dP∗dt). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo−1 hr−1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo−1 hr−1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (fzygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on fzygotic and dP∗dt, the rate of elongation of nascent histone polypeptide chains (At) during the first 6 hr of development was estimated; At remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

Effect of 4.5S RNA depletion on Escherichia coli protein synthesis and secretion.

We examined the synthesis of individual proteins following depletion of 4.5S RNA by using a strain deficient in the induction of heat shock proteins. We found that initially the synthesis of all proteins was equally affected, and the peptide elongation rate was reduced by approximately 10%. For up to 1 generation time after the onset of inhibition of total protein synthesis, the processing of secreted proteins was unaffected. After further depletion of 4.5S RNA, accumulation of precursors of secreted proteins was observed under some growth conditions.

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation in Escherichia coli.

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribing lacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age and restricted feeding on polypeptide chain assembly kinetics in liver protein synthesis in vivo

Polypeptide assembly rates during in vivo hepatic protein synthesis were studied as a function of age and restricted feeding in male rats. With ageing the time to assemble the average peptide in the liver of fully-fed rats significantly increased. In young rats maintained on a restricted feeding regime known to retard ageing, the time to assemble the average polypeptide was increased 2.5 times. With ageing the rate of peptide elongation increased so that at 2 years of age the underfed animals assembled peptides at a significantly faster rate than their age-matched controls. The rate of elongation of peptides during hepatic protein synthesis was shown to be directly dependent upon circulating T 3 levels rather than the dietary status of the animal. On refeeding young diet restricted rats, polypeptide assembly kinetics did not immediately return to control values although the rate of protein synthesis was significantly increased. Total liver RNA content increased significantly in refed animasl allowing for a greater rate of chain intiation to offset the slow rate of chain elongation. A period of 28 days of ad libitum feeding was required before assembly kinetics returned to control values and is probably indicative of a persistent impaired monodeiodination of T 4 to T 3.

Isolation and characterization of an estrogen-regulated ribosome-associated inactivator of tRNA aminoacylation in the uterus

Estradiol (E 2) induces an increase in the peptide elongation rate of isolated uterine ribosomes assayed in a cell-free protein synthesis system. An inhibitory factor, extracted from ribosomes of E 2-deprived rats, was found to inhibit the peptide elongation reaction by acting on certain tRNAs to render them incapable of binding to aminoacyl-tRNA synthetases, thus reducing the availability of specific aminoacylated tRNAs required for the sequential translation of the codons in mRNA. The uterine ribosome-associated tRNA inactivator (RATI) has been partially purified and monoclonal antibodies (MABs) to RATI have been prepared. Specificity of the MABs for RATI was indicated by the inactivation of RATI in vitro by the anti-RATI MABs. RATI selectively inactivates deacylated, but not acylated, tRNAs and the inactivation does not appear to involve nuclease cleavage of the tRNA. Within 1 h after E 2 treatment 50% of both RATI activity and immunoreactivity were lost from the uterine ribosome extracts, suggesting that E 2 regulation of tRNA reutilization may occur through dissociation of RATI from the ribosomal site of tRNA deacylation or alteration in the structure of RATI resulting in inactivation both biologically and immunologically. We propose that RATI may function as an E 2-regulatable ‘witch’ mechanism which inactivates, delays or defers the aminoacylation of certain tRNAs in the absence of E 2 and which participates in the regulation of protein synthesis at the translational level by creating rate-limiting levels of certain tRNAs in the E 2-deprived uterus.

Translational regulation in sea urchin eggs: A complex interaction of biochemical and physiological regulatory mechanisms

AbstractThe unfertilized sea urchin egg is a metabolically quiescent cell. Fertilization results in the activation of a variety of metabolic and biosynthetic pathways, including a 20‐ to 40‐fold increase in the rate of protein synthesis by 2 h after fertilization. This increase is regulated at a purely translational level without the need for new transcription. The greatest part of this increase is due to the translation of stored maternal mRNAs which were not translated in the egg. There is also a 2‐to 3‐fold increase in the peptide elongation rate. The molecular and physiological mechanisms responsible for this activation process are beginning to be understood, and turn out to be much more complex than was anticipated.