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Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.

Kopeina GS, Afonina ZA, Gromova KV, Shirokov VA, Vasiliev VD, Spirin AS - Nucleic Acids Res. (2008)

Bottom Line: The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases.A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate.Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

ABSTRACT
The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

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Sucrose gradient sedimentation profiles of polysomes formed in the wheat germ CECF system with luciferase mRNA. Fifty microliters aliquots of the reaction mixture were taken at the indicated incubation time and analyzed in concave 15–50% sucrose gradient (see ‘Materials and Methods’ section). Gradient zones containing oligosomes (o), medium size (m) and heavy-loaded (h) polysomes are indicated by dashed lines. Optical densities in polysome-containing zones were integrated with IGOR Pro® package. In the presented sedimentation profiles the medium-size polysomes constituted 25, 44, 49, 43, 36, 35%, and heavy-loaded polysomes 5, 19, 24, 35, 42, 53% of total polysome amount at the 10th, 20th, 40th, 60th, 120th and 240th min of incubation time, respectively.
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Figure 3: Sucrose gradient sedimentation profiles of polysomes formed in the wheat germ CECF system with luciferase mRNA. Fifty microliters aliquots of the reaction mixture were taken at the indicated incubation time and analyzed in concave 15–50% sucrose gradient (see ‘Materials and Methods’ section). Gradient zones containing oligosomes (o), medium size (m) and heavy-loaded (h) polysomes are indicated by dashed lines. Optical densities in polysome-containing zones were integrated with IGOR Pro® package. In the presented sedimentation profiles the medium-size polysomes constituted 25, 44, 49, 43, 36, 35%, and heavy-loaded polysomes 5, 19, 24, 35, 42, 53% of total polysome amount at the 10th, 20th, 40th, 60th, 120th and 240th min of incubation time, respectively.

Mentions: The polysome profiles at different time points of the luciferase synthesis in the wheat germ CECF system was analyzed by sucrose gradient centrifugation. Fifty microliters aliquots of translation mixture were withdrawn from the CECF reactor at indicated time points, the reaction was stopped by addition of cycloheximide, and the samples were layered onto sucrose gradients. A concave 15–50% sucrose gradient and short centrifugation time were employed in order to detect the heavy polysome species (see ‘Materials and Methods’ section). Figure 3 shows that after 10 min translation, when the first round of translation was completed, mainly short oligosomes were detected. During the following 30 min the fraction of medium size polysomes (6–12 ribosomes) increased and heavy polysomes containing more than 12 ribosomes began to appear. After 60 min of translation 12 peaks of short and medium size polysomes could be clearly distinguished, and the fraction of heavy polysomes accumulated in lower fractions significantly increased. During subsequent hours of translation the heavy polysomes became a major fraction while the amount of medium size polysomes decreased. The rate of luciferase synthesis remained constant during the entire reaction (Figure 2).Figure 3.


Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.

Kopeina GS, Afonina ZA, Gromova KV, Shirokov VA, Vasiliev VD, Spirin AS - Nucleic Acids Res. (2008)

Sucrose gradient sedimentation profiles of polysomes formed in the wheat germ CECF system with luciferase mRNA. Fifty microliters aliquots of the reaction mixture were taken at the indicated incubation time and analyzed in concave 15–50% sucrose gradient (see ‘Materials and Methods’ section). Gradient zones containing oligosomes (o), medium size (m) and heavy-loaded (h) polysomes are indicated by dashed lines. Optical densities in polysome-containing zones were integrated with IGOR Pro® package. In the presented sedimentation profiles the medium-size polysomes constituted 25, 44, 49, 43, 36, 35%, and heavy-loaded polysomes 5, 19, 24, 35, 42, 53% of total polysome amount at the 10th, 20th, 40th, 60th, 120th and 240th min of incubation time, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377419&req=5

Figure 3: Sucrose gradient sedimentation profiles of polysomes formed in the wheat germ CECF system with luciferase mRNA. Fifty microliters aliquots of the reaction mixture were taken at the indicated incubation time and analyzed in concave 15–50% sucrose gradient (see ‘Materials and Methods’ section). Gradient zones containing oligosomes (o), medium size (m) and heavy-loaded (h) polysomes are indicated by dashed lines. Optical densities in polysome-containing zones were integrated with IGOR Pro® package. In the presented sedimentation profiles the medium-size polysomes constituted 25, 44, 49, 43, 36, 35%, and heavy-loaded polysomes 5, 19, 24, 35, 42, 53% of total polysome amount at the 10th, 20th, 40th, 60th, 120th and 240th min of incubation time, respectively.
Mentions: The polysome profiles at different time points of the luciferase synthesis in the wheat germ CECF system was analyzed by sucrose gradient centrifugation. Fifty microliters aliquots of translation mixture were withdrawn from the CECF reactor at indicated time points, the reaction was stopped by addition of cycloheximide, and the samples were layered onto sucrose gradients. A concave 15–50% sucrose gradient and short centrifugation time were employed in order to detect the heavy polysome species (see ‘Materials and Methods’ section). Figure 3 shows that after 10 min translation, when the first round of translation was completed, mainly short oligosomes were detected. During the following 30 min the fraction of medium size polysomes (6–12 ribosomes) increased and heavy polysomes containing more than 12 ribosomes began to appear. After 60 min of translation 12 peaks of short and medium size polysomes could be clearly distinguished, and the fraction of heavy polysomes accumulated in lower fractions significantly increased. During subsequent hours of translation the heavy polysomes became a major fraction while the amount of medium size polysomes decreased. The rate of luciferase synthesis remained constant during the entire reaction (Figure 2).Figure 3.

Bottom Line: The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases.A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate.Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

ABSTRACT
The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

Show MeSH