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02:33
Initiation of Translation
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2020
Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs. First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
01:44
Termination of Translation
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2020
The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
02:07
Improving Translational Accuracy
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2020
Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
01:37
Translation
0 Views •
2023
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.Translation Produces the Building Blocks of LifeProteins are called the...
01:31
Translation
0 Views •
2023
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation. Translation Produces the Building Blocks of Life Proteins are called the...
FAQs
DNase I first creates random single-strand breaks in double-stranded DNA, exposing 3′-hydroxyl groups at the nicked sites. DNA polymerase I then extends from these starting points. At the same time, its 5′-to-3′ exonuclease activity removes nucleotides ahead of the growing strand, allowing synthesis to replace the displaced DNA rather than simply add material.
Labeled dNTPs serve as substrates as DNA polymerase I synthesizes DNA across regions opened by exonucleolytic removal. Their incorporation marks the newly produced DNA segments, creating a probe that can subsequently be detected through hybridization. Labeling is therefore coupled directly to strand replacement, making the detectable signal part of the DNA used to identify complementary nucleic acid sequences.
Random nicks distribute initiation sites throughout the double-stranded template, giving DNA polymerase I multiple exposed 3′-hydroxyl groups from which to work. Because the polymerase removes nucleotides ahead of synthesis, each site supports localized replacement and label incorporation. This coordinated arrangement is central to generating labeled DNA fragments suitable for subsequent hybridization-based detection.
A typical workflow begins with double-stranded DNA, DNase I, DNA polymerase I, and labeled deoxynucleoside triphosphates. DNase I introduces random nicks, then polymerase I uses the exposed 3′-hydroxyl groups to extend strands while its exonuclease activity removes nucleotides ahead. The labeled nucleotides become incorporated during replacement, producing labeled DNA fragments for use as probes.
The labeled DNA fragments are applied in experiments that allow complementary sequences to hybridize, and the incorporated label provides a way to detect those matches. This supports assays such as Southern blotting and chromosome visualization. Researchers can use the resulting probe signal to identify particular complementary sequences and examine gene location or genome organization.
By revealing where complementary nucleic acid sequences occur, labeled fragments can help analyze genome organization and gene location. In Southern blotting, they support detection of matching DNA sequences, while chromosome visualization uses the labeling principle to locate sequences in a chromosome context. The resulting signal connects molecular sequence complementarity with a genomic or spatial interpretation.