Biology And Biomedical Sciences BIOL 2960 Lecture Notes - Lecture 28: Restriction Enzyme, Aminoacyl Trna Synthetase, Ef-Tu
14 Aug 2018
School
Department
Bio2960 Spring 2018
Study Guide 11: Bacterial Translation, and Recombinant DNA (Lectures 27-28)
Foundational Vocabulary:
At the end of this unit, you should be able to define and/or use the following terms and concepts:
Bacterial Translation:
• Key structures: mRNA, codon, tRNA,
anticodon, inosine, “wobble position”,
polysome
o Ribosome: rRNA, protein, small (30S)
subunit, large (50S) subunit, 16S rRNA,
A (aminoacyl), P (peptidyl) and E (exit)
sites
• Key processes of translation: initiation,
elongation, termination, molecular mimicry
o tRNA Charging: Aminoacyl-tRNA
synthetases, charged tRNA
o Initiation/Ribosome Assembly: fMet
(formylmethionine), initiator tRNA, start
codon (AUG), Shine Delgarno sequence
(= Ribosome Binding Site of mRNA),
Initiation factor IF2, GTPase, 30S
initiation complex, 70S initiation complex
o Elongation: peptide bond formation,
ribozyme, ribosome translocation,
Elongation factors EF-Tu, EF-G,
ribosome A, P, E sites, amino group
o Termination: Release factor RF1
• Translation regulation: Riboswitch
Recombinant DNA:
• Recombinant chimeric DNA, vector, cargo
(a.k.a. insert), transformation, E. coli
• Vector properties: cargo carrier, high-copy
origin of replication, selectable marker,
multiple cloning site (MCS)
• Cloning Strategies:
o restriction enzymes, recognition
sequence, 3’ “sticky end”, blunt end, 5’
“sticky end”, DNA ligase
o “Cloning by PCR”, primers with
restriction enzyme recognition
sequences, amplification
• DNA libraries: genomic DNA library, cDNA
library, reverse transcriptase, processed
mRNA, cell-type dependency
When you’re done studying for this unit, you should be able to:
20. Outline the steps of translation initiation, elongation, and termination in bacteria, including the tRNA
synthetases, translation factors, tRNAs, and rRNA active sites.
20.1. Recall the role of the following RNAs during translation: tRNA, rRNA, mRNA.
20.2. Determine which factor “knows” the genetic code and which factor establishes the reading
frame.
20.3. When given the number of Shine-Delgarno sequences present on a prokaryotic transcript,
predict the number of unique proteins translated from that transcript.
20.4. Defend the idea that enzymes can be either protein or RNA using an example from translation.
20.5. Explain both how the structure of each of the following factors fits its function in translation and
how the structure of each of the following ensures accuracy or efficiency during translation: (1)
Aminoacyl tRNA synthetase, (2) Initiation Factor 2, (3) Elongation factor G (EF-G), (4)
Elongation Factor Tu (EF-Tu), (5) Release Factor 1 (RF1)
20.6. Identify the GTPases involved in translation and predict the effect of impaired GTPase function
for each.
20.7. Recall whether translation can be “coupled” to transcription in prokaryotes and/or eukaryotes.
20.8. On a diagram that includes multiple ribosomal complexes concurrently translating an mRNA
(polysome), be able to:
20.8.1.Infer mRNA and amino acid chain directionality,
20.8.2.Infer the relative location of the start codon and the Shine-Delgarno sequence
20.8.3.Draw and label the component factors of the initiation complex, including relevant RNA
sequences
20.8.4.Infer the direction of ribosomal movement.
20.9. Identify one example of proofreading during the process of translation.
