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Related Concept Videos

Improving Translational Accuracy02:07

Improving Translational Accuracy

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...
Improving Translational Accuracy02:07

Improving Translational Accuracy

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...
Leaky Scanning02:28

Leaky Scanning

During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Initiation of Translation02:33

Initiation of Translation

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...
tRNA Activation02:26

tRNA Activation

Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...

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Hyperaccurate and error-prone ribosomes exploit distinct mechanisms during tRNA selection.

Hani S Zaher1, Rachel Green

  • 1Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Molecular Cell
|July 7, 2010
PubMed
Summary

Restrictive and ribosomal ambiguity mutations in Escherichia coli ribosomes alter tRNA selection by affecting near-cognate tRNA off-rates. These mutations impact distinct phases: proofreading for restrictive and initial selection for ribosomal ambiguity, suggesting independent functions of ribosomal subunit regions.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Escherichia coli strains with hyperaccurate (restrictive) and ribosomal ambiguity (ram) phenotypes are linked to mutations in rpsL and rpsD/rpsE genes.
  • Ribosomal proteins S12 and S4/S5, encoded by these genes, are located in distinct regions of the small ribosomal subunit crucial for domain closure during tRNA selection.

Purpose of the Study:

  • To investigate the impact of rpsL and rpsD mutations on the distinct phases of tRNA selection: initial selection and proofreading.
  • To elucidate the mechanistic basis of restrictive and ram phenotypes in bacterial translation fidelity.

Main Methods:

  • Analysis of restrictive and ram Escherichia coli ribosomal mutants.
  • Characterization of tRNA selection dynamics, focusing on off-rates of near-cognate tRNAs.
  • Utilizing crystallographic data of ribosomal proteins S12, S4, and S5.

Main Results:

  • Both restrictive and ram ribosomes primarily influence tRNA selection by altering the off-rates of near-cognate tRNA species.
  • Restrictive ribosomes predominantly affect the proofreading phase of tRNA selection.
  • Ram ribosomes primarily impact the initial selection phase of tRNA selection.

Conclusions:

  • The study reveals that distinct regions of the small ribosomal subunit, involving S12/h27/h44 and S4/S5 interfaces, function independently.
  • These independent interfaces contribute to high-fidelity tRNA selection through modulation of distinct kinetic steps.
  • Mutations in rpsL and rpsD differentially affect the kinetic phases of tRNA selection, explaining the observed phenotypes.