Lesson 7: Translation

What does AUG (start codon) code for?

Methionine

How many tRNA’s must each cell contain at a minimum?

20 (one for every amino acid)

Why does tRNA have some modified bases?

for stabalizatrion, flexability, and wobble-pairing

What is the Acceptor stem?

amino acid becomes covalently attached to tRNA at the 3’ end of this stem

What is the Anticodon arm?

contains the anticodon, a three-base sequence that binds to a complementary codon in mRNA

What is the TyC arm?

contains thymidylate (T) and pseudouridylate (y) followed by C

What is the D arm?

contains dihydrouridylate (D)

What is the Variable arm?

ranges from 3-21 nucleotides

How are tRNA named?

named for the amino acid that they carry (e.g. tRNAPhe)

What is the “wobble” position?

the 5’ anticodon position has some flexibility in base pairing; 3rd base of an mRNA codon (and the corresponding 1st base of the tRNA anticodon) where base-pairing is flexible.

What is the Isoacceptor tRNA molecules?

different tRNA molecules that bind the same amino acids

What is the purpose of the wobble?

allows some tRNA molecules to recognize more than one codon

How are Isoacceptor tRNAs identified?

Roman numerals or codons: tRNAI Ala, tRNAII Ala or tRNAGCG Ala

How many different tRNA do Bacteria have?

30-60 different tRNA

How many differnt tRNAs do Eukaryotes have?

up to 80 different tRNAs

What is Aminoacyl-tRNA?

amino acids are covalently attached to the 3’ end of each tRNA molecule

How are Aminoacyl-tRNA named?

alanyl-tRNA^Ala

The activation of an amino acid by aminoacyl-tRNA synthetase requires what?

ATP

What enzyme activates amino acids?

aminoacyl-tRNA synthetase

What direction does the ribosome complex move along the template mRNA?

5’ → 3’

What direction is polypeptide synthesized in?

N (‘5) → C (3’)

All ribosomes contain ______ subunits of _____size

two, unequal

When is the ribosomal complex assembled?

start of mRNA coding sequence

When is the ribosomal complex terminated?

end of mRNA coding sequence

What does the Ribosomal complex consist of?

1. ribosomal subunit

2. mRNA template to be translated

3. initiator tRNA

4. protein initation factors

Each cell contains at least two methionyl-tRNAMet molecules which recognize AUG, these are?

• initiator tRNA

• tRNA^Met

What does the initiator tRNA recongnize?

initiation codons

What does the tRNA^Met recongnize?

only internal AUG

What is the initiator tRNA in bacteria?

N-formylmethionyl-tRNAf Met

What is the initiator tRNA in Eukaryotes?

methionyl-tRNAi Met

What does N-formylmethionyl-tRNAf^Met have that methionyl-tRNAi^Met does not?

an extra carboxide group

In prokaryotes, the 30S ribosome binds to what to start translation?

Shine Dalgarno sequence

What else does the Shine Dalgarno sequence bind to?

a complementary base sequence at the 3’ end of the 16S rRNA

Different between the naming of the Prokaryotes vs Eukaryotes Initiation factors?

the letter “e” (IF-1 vs eIFs)

What are Initiation factors?

help form the initaition complex

What does Eukaryotic initiation factor 4 (eIF-4), (or cap binding protein, CBP) do?

binds to the (5’ end) 7methylguanylate cap of eukaryotic mRNA

The initiator tRNA is in the which site?

P site

Which site is ready to receive an aminoacyl-tRNA?

A site

What is step 1 of elongation?

Positioning the correct aa-tRNA in site A

What is step 2 of elongation?

Formation of a peptide bond

What is step 3 of elongation?

Shifting mRNA by one codon

How many phosphoanhydride bonds are cleaved for each amino acid added to a polypeptide chain?

4

How many P bonds are broken (ATP used) for amino acid activation?

2

How many P bonds are broken (ATP used) for elongation?

2

How many P bonds are used for 100 amino acids?

100 amino acid = 99 bonds

99 × 4 = 396 broken

How many ATP/GTP are used for translation?

1 ATP (= 2 P) for activation and 2 GTP (= 2GDP + 2P) for elongation

What can Puromycin do?

An antibiotic that resembles the 3’ end of an aminoacyl-tRNA, and can enter the A site of a ribosome. The peptidyl-puromycin formed is bound weakly in the A site and dissociates, terminating protein synthesis

Ribosomal Protein Synthesis Is Coupled to what in E. coli?

Ribosome Assembly

When elF-2 (elongation factor II) is phosphorylated, what happens?

protein syntheiss is inhibited

What does elF-2 do?

binds GTP + initiator tRNA (Met-tRNAi), forming a complex and delivers tRNA to the ribosome → translation begins

When DNA interacts with Polymerase I, what is produced?

rRNA

When DNA interacts with Polymerase II, what is produced?

mRNA

When DNA interacts with Polymerase III, what is produced?

tRNA

What is the A site?

aminoacyl site

What is the P site?

Peptidyl site

What is the E site?

exit site

What is the function of the A site?

Where charged tRNAs enter the ribosome

What is the funtion of the P site?

Holds onto the growing peptide chain (also holds 1st amino acid: Met)

What is the function of the E site?

Where tRNAs are ejected from the ribosome after their amino acid has been incorporated into the growing peptide chain

What ribosomal subunit are A, P, E sites located?

large, 60S subunit

What ribosomal subunit is the mRNA binding site located?

small, 40S subunit

What is the Wobble?

Nontraditional base pairing between the 5ʹ nucleotide (1st nucleotide) of the anticodon and the 3ʹ nucleotide (3rd nucleotide) of the codon. → this allows same tRNA to translate more than one codon as long as the amino acid is the same

What is an example of the wobble effect?

How many tRNA are required for 61 codons because of genetic code redundancy?

32

In eukaryotic translation initiation, 5’ cap is directly connected to what?

40S subunit

In prokaryotic translation initiation, shrine-dalgarno is directly connected to what?

30S subunit

What do Pseudomonas and diphtheria toxin inhibit?

ADP-ribosylation of eEF-2

What do shiga toxins inhibit?

Aminoacyl-tRNA binding to A site

What do shiga toxins do?

cuts 28s rRNA on 60S Eukaryotic subunit

What two Aminoglycosides are involved in prokaryotic translation?

Steptomycin and Gentamycin

What effect do the two Aminoglycosides have in prokaryotic translation?

indirectly inhibit translation by cause misreading of mRNA or interupting translation initation

What part of the ribosome comlex is indirectly impacted by the two aminoglycosides?

30S subunit

What is Linezolid?

antibiotic that targets the 50S ribosomal subunit

What interferes with the 16S rRNA of the 30S ribosomal subunit interfering with Shine Delgerno?

Aminoglycosides

What are macrolides?

antibiotics that target the bacterial ribosome (50S) and interfere with translation elongation

What does Clindamycin do?

interferes with the peptidyl transferase center and disrupts movement of the ribosome along mRNA, freezes elongation on the 50S subunit

What does tetracycline do?

antibiotic that targets the 30S ribosomal subunit and blocks aminoacyl-tRNA from binding to the A site so no new amino acid can be added to the growing peptide chain

What does Chloramphenicol do?

an antibiotic that targets the 50S ribosomal subunit and inhibits the peptidyl transferase enzyme preventing formation of peptide bonds between amino acids or preventing them from linking together

eEF-2 (Eu) is a what?

G-protein

What is Gray Baby syndrome?

a serious and potentially fatal condition in newborns caused by chloramphenicol toxicity.

What are 3 factors of I-Cell disease?

1. Fibroblasts

2. Mannose-6-phosphate.

3. Coarse facial features

What are Fibroblasts?

Very common to find the presence of inclusion bodies in fibroblasts

What is Mannose-6-phosphate?

Before going to the lysosome, enzymes must be tagged by M6P, I-cell disease will not => large inclusions from molecules that should’ve been degraded by the lysosome and are just sitting there … forever waiting

What are Coarse facial features?

add to that joint pain and psychomotor retardation in a young child

What is i-Cell disease?

defect in phosphotransferase

Where does transcription occur in Eukaryotes?

Nucleus

Where does transcription occur in Prokaryotes?

Cytoplasm

Where does translation occur in prokaryotes?

cytoplasm, on ribosome (somtimes on RER)

Where does translation occur in Eukaryotes?

cytoplasm, on ribosome (somtimes on RER)

Where does the protein go after translation?

Golgi → vesicle → final destination

What happens in the Golgi?

other sugars are added

What are heat shock proteins?

a type of chaperone proteins, used to help fold complex proteins

What are the Enhancer region response elements?

GRE = glucucoid response element

CRE = cGMP response element

ERE = estrogen response element

Harmones bind to the response elements and cause the DNA to?

bend so its easier to transcribe

Peroxisome proloferator activated receptors (PPARs) (alpha and gamma)

Harmone response element regulates multiple aspects of lipid metabolism

activated by fibrates and thiazolidinediones

fibrates effect

acts on PPAR-alpha and lowers TGs

thiazolidinediones effect

acts on PPAR-gamma and lowers TGs

cyclooxygenage II is located downstream of what?

NFkB (nuclear factor kappa-B)

What is NFkB (nuclear factor kappa-B) involved in?

Regulates expression of many genes in the immune system and is involved in inflamation

Glucocorticoids effect

decreases signaling because decreased kB element binding