Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.
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Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA. / Kopeina, Gelina S; Afonina, Zhanna A; Brune, Kira; Shirokov, Vladimir A; Vasiliev, Victor D; Spirin, Alexander S.
In: NUCLEIC ACIDS RES, Vol. 36, No. 8, 8, 2008, p. 2476-2488.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.
AU - Kopeina, Gelina S
AU - Afonina, Zhanna A
AU - Brune, Kira
AU - Shirokov, Vladimir A
AU - Vasiliev, Victor D
AU - Spirin, Alexander S
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
KW - Kinetics
KW - Centrifugation, Density Gradient
KW - RNA, Messenger/metabolism
KW - Protein Biosynthesis
KW - Cell-Free System
KW - Luminescent Proteins/genetics
KW - 3' Untranslated Regions/chemistry
KW - 5' Untranslated Regions/chemistry
KW - Luciferases/genetics
KW - Polyribosomes/metabolism/ultrastructure
KW - Ribosomes/metabolism
KW - Tobacco Mosaic Virus/genetics
KW - Triticum/genetics
KW - Kinetics
KW - Centrifugation, Density Gradient
KW - RNA, Messenger/metabolism
KW - Protein Biosynthesis
KW - Cell-Free System
KW - Luminescent Proteins/genetics
KW - 3' Untranslated Regions/chemistry
KW - 5' Untranslated Regions/chemistry
KW - Luciferases/genetics
KW - Polyribosomes/metabolism/ultrastructure
KW - Ribosomes/metabolism
KW - Tobacco Mosaic Virus/genetics
KW - Triticum/genetics
M3 - SCORING: Journal article
VL - 36
SP - 2476
EP - 2488
JO - NUCLEIC ACIDS RES
JF - NUCLEIC ACIDS RES
SN - 0305-1048
IS - 8
M1 - 8
ER -
