Lecture 15 - Translation Biomed

_______________ are the end products of the information pathways in the Central Dogma of Molecular Biology

Proteins

TRUE/FALSE

Every prokaryotic and eukaryotic cell contains from several to thousands of copies of many different proteins

True

TRUE/FALSE

Proteins are made at slow rates due to the great complexity of protein synthesis

False

Despite the great complexity of protein synthesis, proteins are made at exceedingly high rates.

How long does it take Escherichia coli to synthesize a polypeptide of 100 resides?

5 seconds

There are ____ major advancements in the field of protein synthesis

Three

The first major advancement in the field of Protein Synthesis involves the identification of the site of protein synthesis by ______________ and his colleagues in 1950s: Ribosome is the site of protein synthesis

Paul Zamecnik

The second major advancement in the field of Protein Synthesis involves the discovery that amino acids were "activated" when incubated with ATP. Discovered by ___________ __________ and _____________.

Mahlon Hoagland and Zamecnik

The third major advancement in the field of Protein Synthesis involves ___________ ______________'s reasoning on how the genetic information encoded in the 4- letter language of nucleic acids could be translated into the language of proteins

Francis Crick

A ___________ is a triplet of nucleotides that codes for a specific amino acid

Codon

TRUE/FALSE

The Genetic Code has no overlap or "punctuation"

True

______________ codon is the first codon that sets the reading frame.

Start/Initiation

What is the start/initiation codon sequence?

AUG

What amino acid does the start/initiation codon code for?

Methionine

TRUE/FALSE

The synthetic machinery reads sequentially from one triplet to the next

True

What do termination codons do?

Signal the end of polypeptide

What are the other names for the termination codons?

Stop codons or nonsense codons

What are the 3 different sequences for the termination codons?

- UAA

- UAG

- UGA

The Genetic Code is __________________: amino acid may be specified by more than one codon

Degenerate

Ex. Amino acid Phenylalanine: UUU, UUC

TRUE/FALSE

Amino acids can have more than one codon

True

TRUE/FALSE

Each codon can specify more than one amino acid

False

Each codon specifies only one amino acid

TRUE/FALSE

The genetic code is completely universal

False

The genetic code is nearly universal

What are the exceptions of a completely universal genetic code?

- Minor variations in mitochondria

- Variations in some bacteria

- Variations in some single-celled eukaryotes

In the genetic code, there is _____________ of the coding nucleotide sequence of DNA, mRNA, and the amino acid sequence of a polypeptide chain.

Colinearity

The triplets of nucleotide units in _________ determine the amino acid sequence in a __________ through the intermediary ________.

- DNA

- Protein

- mRNA

The evidence of DNA coding for amino acids through mRNA is seen through ____________ (Changes in the DNA sequence results in changes in the composition and length of the polypeptide chain)

Mutations

______________ transfers the amino acids to mRNA for synthesis of protein, also called adaptor molecules

Transfer RNA (tRNA)

The three-base sequence on the mRNA that codes for an amino acid is called the _____________, while the three-base sequence on the tRNA is called the ______________.

- Codon

- Anti-codon

TRUE/FALSE

The anticodon triplet of a tRNA recognizes only one codon triplet through Watson-Crick base pairing at all three positions

False

Cells would have a different tRNA for each amino acid codon if tRNA only recognized one codon

What is the total number of codons?

4^3 = 64

What is the number of different tRNA molecules?

~30-40

What does "I" represent in a tRNA base?

Base that can pair/form hydrogen bonds with U, C, and A

What is the hypothesis that describes how tRNA can recognize multiple codons that can encode for the same amino acid?

Wobble Hypothesis

Which bases of the mRNA codon always form strong Watson-Crick base pairs with the corresponding bases of the tRNA anticodon and confer most of the coding specificity?

The first two bases of an mRNA codon

Which base of the anticodon and which base of the codon results in Wobble or less precise base pair?

The first base of the anticodon and the third base of the codon

What is known as the "Protein Factory" where protein synthesis is performed?

Ribosomes

Ribosomes are made up of __________ and __________.

- Proteins

- rRNA

The ________ subunit of a ribosome carries the tRNA for accurate matching of tRNA with the codon

Small

The _________ subunit of a ribosome helps in protein synthesis

Large

What is the composition of a Prokaryotic ribosome?

- Prokaryotes—70S

- Small (30S)-21 proteins + 16S rRNA

- Large (50S)- 34 proteins + 23S & 5S rRNA

What is the composition of a Eukaryotic ribosome?

- Eukaryotes— 80S

- Small (40S)- 33 proteins + 18S rRNA

- Large (60S)- 49 proteins 28S, 5S, 5.8S rRNA

What does the "S" represent in ribosomal subunits?

Sedimentation Rate

What are the 4 steps in Translation?

1. Activation of Amino Acids

2. Initiation

3. Elongation/Translocation

4. Termination

What is the first step in the activation of amino acids?

Linking of amino acid to tRNA

What enzyme catalyzes the addition of amino acid to tRNA?

Aminoacyl-tRNA synthetase

How many Aminoacyl-tRNA synthetases are there for each amino acid?

- One enzyme for each amino acid

- 20 total

Ex. Alanine tRNA synthetase

TRUE/FALSE

Aminoacyl-tRNA synthetases have no proof reading activity

False

Aminoacyl-tRNA synthetases have proof reading activity

When are amino acids considered "charged"?

Once the tRNA are bound to amino acid

What is the first codon in eukaryotes?

What is the first codon in bacteria?

Eukaryotes: AUG: amino acid methionine

Bacteria: A modified form of methionine - formylmethionine - is used

In bacterial translation, what is the leader sequence upstream of the first AUG that helps in the correct positioning of the small subunit of the ribosome?

Shine-Dalgarno Sequence

What is the Shine Dalgarno nucleotide sequence?

5′-AGGAGGU-3′

Is the Shine Dalgarno sequence found in eukaryotes, bacteria, or both?

Bacteria

What binds to the initiation complex to form an intact ribosome in bacteria?

The 50S ribosomal subunit

How many initiation factors are involved in the Initiation step of bacterial translation?

Three initiation factors are involved

Binding of the large subunit (50S) has what 3 sites?

A= Aminoacyl site

P= Peptide Site

E= Exit

Which site of the ribosome has a tRNA carrying the next amino acid in the chain binds to the ribosomal ____site by forming base pairs with the codon in mRNA

A site

Which site of the ribosome has the carboxyl end of the polypeptide chain which is released from the tRNA at the ____site and joined to the free amino group of the amino acid linked to the tRNA at the A-site, forming a new peptide bond

P site

What is the enzyme that catalyzes the reaction at the P site?

Peptidyl transferase

Where does the enzymatic activity reside in the ribosome?

The 23S rRNA

RNA that acts as enzymes are called ______________.

Ribozymes

Which site of the ribosome releases dissociated tRNAs so that the can be recharged with free amino acids?

E site

Elongation proceeds with single-codon movements of the ribosome each called a __________________ event.

Translocation

What are the 2 elongation factors involved in the elongation phase of Translation?

- EF-Tu

- EF-G

The _______________ of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA

Termination

What does the binding of the release factor at the A site do?

- Releases polypeptide from the P site

- Small and large ribosomal subunits dissociate from the mRNA

How does mRNA binding of the small ribosomal subunit occur in Eukaryotes vs Prokaryotes?

- Eukaryotes: 5' cap recognition, scans to first AUG

- Prokaryotes: Shine-Delgarno sequence (purine-rich) indicates correct AUG for start

How does the first amino acid in translation differ from Eukaryotes to Prokaryotes?

- Eukaryotes: First amino acid is Methionine

- Prokaryotes: First amino acid is Formyl-Methionine

How do Initiation Factors differ from Eukaryotes to Prokaryotes?

- Eukaryotes: eIFs (>12)

- Prokaryotes: IFs (3)

- eIF = Eukaryotic initiation factors

- IF = Initiation factors

In eukaryotic translation initiation, Methionyl-tRNA binds to _______ and ________.

- eIF2

- GTP

The Methionyl-tRNA then interacts with the ______ subunit of the ribosome.

Small

___________ complex binds to the 5' Cap of the mRNA strand. (ATP is required)

eIF4

What happens when the ribosome-methionyl-tRNA complex interacts with the cap complex?

Moves along mRNA strand until it finds the start of translation AUG

GTP is hydrolyzed, _______ are released and the ____________ ribosomal subunits binds Methionyl-tRNA binds to ___ site.

- eIFs

- Large

- P site

How many initiation factors are involved in Eukaryotic Translation Initiation?

At least 9 initiation factors are involved

Two elongation factors _____________ and _______________ enter and leave the ribosome during each cycle.

- eEF1A

- eEF2

Aminoacyl-tRNA binds to a complex of GTP-bound __________

eEF1A

The resulting aminoacyl-tRNA-eEF1A-GTP complex binds to the ___ site of initiation complex

A site

___________ bond is now formed between the two amino acids bound by their tRNAs to the ___ and ___ sites. The enzyme that catalyzes peptide bond formation is _____________ _______________.

- Peptide

- A and P sites

- Peptidyl transferase

Elongation factor __________ along with GTP helps to translocate the complex to the next codon: Translocation

eEF2 (Translocase)

New ____ site results, Peptidyl-tRNA moves to ___ site, Free t-RNA (Uncharged) moves to ____ site and dissociates.

- A site

- P site

- E site

Translation termination in eukaryotes occurs with the encounter of a _______ codon

Stop

What are the termination factors in eukaryotes that assist in termination?

eRF1 and eRF3 assist in termination

During termination, protein is released along with _________ and ___________. Ribosome dissociates into ___ subunits.

- tRNA

- mRNA

- 2

What antibiotics inhibit Prokaryotic Translation?

- Puromycin

- Streptomycin

- Tetracyclines

__________________ inhibits initiation and misreading of the genetic code

Streptomycin

_________________ causes premature chain termination by acting as an analog of aminoacyl-tRNA and does not engage in translocation

Puromycin

__________________ blocks the A (inhibiting the 30S ribosomal units) and preventing aminoacyl-tRNA binding for protein synthesis

Tetracyclines

What inhibits protein synthesis in Eukaryotes?

Diphtheria toxin

The Diphtheria toxin inhibits _______________.

Translocation

How does Diphtheria toxin inhibit eukaryotic translation?

- A fragment catalyzes the ADP-ribosylation of eEF2 blocking its capacity to act in translocation of polypeptide chain being synthesized

- Protein synthesis is completely inhibited

What are 2 protein synthesis inhibitors in dentistry?

1. Clindamycin

2. Tetracylines

What does Clindamycin do?

A very common antibiotic used in dentistry and works by inhibiting protein synthesis. Prevents peptide bond formation, inhibiting protein synthesis by reversibly binding to 50S ribosomal subunits.

What is the use of Clindamycin in dentistry?

It is the drug of choice if one has a penicillin allergy. Tooth infections.

What do tetracyclines do?

Inhibit the 30S ribosomal subunit, hindering the binding of the aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex

What is the use of Tetracycline in dentistry?

Tetracycline periodontal fibers are used to help treat periodontal disease (a disease of gums). Periodontal disease is caused by bacteria growing beneath the gum line.

Can transcription and translation be a coupled/synchronous process in prokaryotes?

Yes

Can transcription and translation be a coupled/synchronous process in eukaryotes?

- No

- Transcription and translation are not coupled/ asynchronous process