Protein Synthesis 2
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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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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

  • 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


Protein synthesis 2 major topics covered translation initiation elongation and termination

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”.


Protein synthesis 2 major topics covered translation initiation elongation and termination

I will start with a general overview of translation (the example is eukaryotic)


Protein synthesis 2 major topics covered translation initiation elongation and termination

The ribosome is the ribozyme that catalyzes peptide bond formation.

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

A general cartoon of the translation process


Protein synthesis 2 major topics covered translation initiation elongation and termination

So, where does this start?

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

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).


Protein synthesis 2 major topics covered translation initiation elongation and termination

Three translation initiation factors are required (in addition to the ribosome and aa-tRNA)


Protein synthesis 2 major topics covered translation initiation elongation and termination

The process of translation initiation in

prokaryotic cells


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

The translation elongation cycle


Protein synthesis 2 major topics covered translation initiation elongation and termination

The chemistry of peptide bond formation


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

Important term = molecular mimicry

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

From Ramakrishnan, Cell 108: 557 (2002)


Protein synthesis 2 major topics covered translation initiation elongation and termination

Translation is a cycle

(final overview)


Protein synthesis 2 major topics covered translation initiation elongation and termination

A more detailed animation of translation, including factors


Protein synthesis 2 major topics covered translation initiation elongation and termination

Translation is a highly conserved process among all living things…

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

Important difference #1:

Ribosomes are substantially different


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

The structural arrangement and required factors for translation initiation are substantially different in eukaryotes, compared to bacteria


Protein synthesis 2 major topics covered translation initiation elongation and termination

Overview of eukaryotic translation initiation


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

Translation elongation is very similar in eukaryotes and prokaryotes

eEF1a

eEF1b

eEF2


Protein synthesis 2 major topics covered translation initiation elongation and termination

Translation termination in eukaryotes is mechanistically similar to prokaryotes…

Important difference:

only one release factor is required


Protein synthesis 2 major topics covered translation initiation elongation and termination

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.


Protein synthesis 2 major topics covered translation initiation elongation and termination

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

Why does this matter?


Protein synthesis 2 major topics covered translation initiation elongation and termination

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!


Protein synthesis 2 major topics covered translation initiation elongation and termination

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

Puromycin is a charged tRNA (tRNATyr) analog:

as expected it inhibits translation in all organisms


Protein synthesis 2 major topics covered translation initiation elongation and termination

Many human genetic disorders originate from nonsense mutations

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

Many human genetic disorders originate from nonsense mutations

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


Protein synthesis 2 major topics covered translation initiation elongation and termination

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

-Kerem, et al. The Lancet (2008)


Protein synthesis 2 major topics covered translation initiation elongation and termination

THE END

-any questions?


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