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Protein Synthesis 2 Major topics covered: Translation: initiation, elongation and termination

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Protein Synthesis 2 Major topics covered: Translation: initiation, elongation and termination Comparison of eukaryotic translation to prokaryotic Medical relevance of translation: two points . c ontact info: David A. Schneider, Ph.D. Department of Biochemistry and Molecular Genetics

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Presentation Transcript
slide1
Protein Synthesis 2
  • Major topics covered:
  • Translation: initiation, elongation and termination
  • Comparison of eukaryotic translation to prokaryotic
  • Medical relevance of translation: two points

contact info:

David A. Schneider, Ph.D.

Department of Biochemistry and Molecular Genetics

[email protected]

office #: 934-4781

related text:

Biochemistry

Garret and Grisham, 4th ed.

Chapter 30

slide2
Reminder of Friday’s lecture:
  • Translation is the process of making protein from an RNA template
  • Fidelity is affected by several steps
  • tRNAs are the “adapters” that translate the 4-nucleotide language of DNA/RNA into the 20-amino acid language of proteins.
  • Aminoacyl-tRNAsynthetases are ancient, but accurate enzymes.
  • Ribosomes are large, complicated “machines”.
slide4
The ribosome is the ribozyme that catalyzes peptide bond formation.

What other factors participate in translation, and how is the whole process orchestrated?

slide5
The ribosome is the ribozyme that catalyzes peptide bond formation.

What other factors participate in translation, and how is the whole process orchestrated?

  • Translation consists of three steps:
            • Initiation
            • Elongation
            • Termination
slide7
So, where does this start?

In bacteria, the first codon in the mRNA (AUG) leads to initiation and recruitment of the formyl-methionyltRNA

slide8
How does the ribosome find the first codon?

The “Shine-Delgarno” sequence in the mRNA:

Base pairing between the Shine Dalgarno sequence and the 3´ end of 16S rRNA facilitates translation initiation. Consequently, the efficiency of translation initiation is determined by:

1) How well the S.D. sequence conforms to the consensus sequence that is complementary to the 3´ end of 16S rRNA.

2) The distance between the S.D. sequence and the start codon (a 7 base spacer is optimal).

slide9
Three translation initiation factors are required (in addition to the ribosome and aa-tRNA)
slide11
High translation initiation rates lead to multiple ribosomes per message (“polysomes”)

Electron micrograph of polysomal mRNA

Note of interest: ribosome occupancy on mRNA plays a major role in determining mRNA decay rate

slide14
Translation terminates when a stop codon (UAA, UAG, UGA) enters the A-site

Translation termination factors:

RF-1 = recognizes UAA and UAG

RF-2 = recognizes UAA and UGA

RF-3 = G-protein; helps trigger hydrolysis

(by the 23S rRNA)

RRF = liberates ribosome/release factors

slide15
Important term = molecular mimicry

Translation factors use molecular mimicry to utilize common binding sites on the ribosome

From Ramakrishnan, Cell 108: 557 (2002)

slide16
Translation is a cycle

(final overview)

slide18
Translation is a highly conserved process among all living things…

However, important differences exist between bacteria and eukaryotes (e.g. you!)

slide19
Important difference #1:

Ribosomes are substantially different

slide20
Important difference #2: mRNA is very different in prokaryotes versus eukaryotes

Bacterial mRNA: lacks 5’ cap, poly-A tail not required, multiple orfs per transcript, SD sequence

eukaryotic mRNA: 7-MeG cap, poly-A tail, one orf per transcript, no sequence specific binding

Consequence: translation mechanisms are different, primarily at the initiation step

slide21
The structural arrangement and required factors for translation initiation are substantially different in eukaryotes, compared to bacteria
slide23
Step 1: eIF1, 1A, 3 and 5 bind to 40S (not shown)

tRNAiMet-eIF2:GTP is recruited

Step 2: eIF4 proteins associate with mRNA (cap and tail) and bind 43S preinitiation complex

Scanning

Step 3: eIF5-mediated ejection of IFs and 60S binds

slide25
Translation termination in eukaryotes is mechanistically similar to prokaryotes…

Important difference:

only one release factor is required

slide26
What have we learned (lectures 1&2)?
  • tRNAs “adapt” the 4-base nucleotide code to a 20 amino acid protein code.
  • Charging of tRNAs and codon:anticodon interactions are critical for fidelity.
  • Ribsomes are big-big-big ribozymes… that we can now visualize in some detail.
  • Translation is a complicated process that is geared to be efficient and accurate!
  • Eukaryotic translation varies from prokaryotic translation most significantly at the initiation step.
slide27
We know that translation and ribosome composition varies between bacteria and eukaryotic cells

Why does this matter?

slide28
We know that translation and ribosome composition varies between bacteria and eukaryotic cells

Why does this matter?

Fungi and bacteria often occupy the same environment and battle for the same resources. Thus, they try to kill each other

We benefit!

slide29
Several common antibiotics with mode of action and molecular target (of some) mapped

Note: your mitochondrial ribosomes are similar to those of bacteria,

thus some toxicity occurs

slide30
Puromycin is a charged tRNA (tRNATyr) analog:

as expected it inhibits translation in all organisms

slide31
Many human genetic disorders originate from nonsense mutations

Nonsense mutations: premature stop codons in orf leading to termination of translation and incompletely synthesized protein

slide32
Many human genetic disorders originate from nonsense mutations

Nonsense mutations: premature stop codons in orf leading to termination of translation and incompletely synthesized protein

slide33
PTC124 has progressed effectively through Phase 2 clinical trials and can rescue CFTR mRNA levels

-Kerem, et al. The Lancet (2008)

slide34
THE END

-any questions?

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