Download MBBS Biochemistry PPT 27 Translation Lecture Notes

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? Objectives

? Mutation
? Steps of Protein synthesis

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? Prokaryotes
? Eukaryotes

? Post translational Modification
? Clinical implications

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Case 1

? A 3-year-old Caucasian boy is brought to the clinic for a chronic

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productive cough not responding to antibiotics given recently. He

has no fever or sick contacts. His medical history is significant for

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abdominal distention, failure to pass stool, and emesis as an infant.

He continues to have bulky, foul-smel ing stools. No diarrhea is

present. He has several relatives with chronic lung and "stomach"

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problems, and some have even died at a young age. The

examination reveals an il appearing, slender male in moderate

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distress. The lung exam reveals poor air movement in the base of

lungs bilateral and coarse rhonchi throughout both lung fields. A

chloride sweat test was performed and was positive, indicating

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cystic fibrosis (CF).

? What is the mechanism of the disease?

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? How might gel electrophoresis assist in making the diagnosis

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Case 2

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? An 8-year-old boy is brought to his pediatrician by his

mother because she was concerned that he was having

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language-speech problems, was hyperactive, and was told

by teachers that he may have mental retardation. The

mother reports a strong family history of mental

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retardation in males. The boy on exam is found to have a

large jaw, prominent ears, and enlarged testes

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(macroorchidism). The mother was told her family had a

genetic problem causing the mental retardation. The

patient underwent a series of blood tests and was

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scheduled to see a genetic counselor, who expressed that

the etiology of the genetic defect was likely transmitted

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from his mother. The genetic counselor states that his

mother likely has a silent mutation.

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? What is the most likely diagnosis?
? Which chromosome is likely to be

affected?

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? What are some types of biochemical

mutations?

? What is the biochemical basis of the

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different types of mutations?

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Case 3

? A 15-year-old African-American female presents to the emergency

room with complaints of bilateral thigh and hip pain. The pain has

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been present for 1 day and is steadily increasing in severity.

Acetaminophen and ibuprofen have not relieved her symptoms.

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She denies any recent trauma or excessive exercise. She does report

feeling fatigued and has been having burning with urination along

with urinating frequently. She reports having similar pain episodes

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in the past, sometimes requiring hospitalization. On examination,

she is afebrile (without fever) and in no acute distress. No one in

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her family has similar episodes. Her conjunctiva and mucosal

membranes are slightly pale in coloration. She has nonspecific

bilateral anterior thigh pain with no abnormalities appreciated. The

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remainder of her examination is completely normal. Her white

blood cel count is elevated at 17,000/mm3, and her hemoglobin

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(Hb) level is decreased at 7.1 g/dL. The urinalysis demonstrated an

abnormal number of numerous bacteria.

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? What is the most likely diagnosis?
? What is the molecular genetics behind this

disorder?

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? What is the pathophysiologic mechanism

of her symptoms?

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Sickle cel disease (pain crisis).

? CLINICAL CORRELATION

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? This 15-year-old female's description of her pain is typical of a sickle cell

pain crisis.

? infection is a trigger, most commonly pneumonia or a urinary tract

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infection. This case is consistent with a urinary tract infection, indicated by

her symptoms of urinary frequency, and burning with urination (dysuria).

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? Her white blood cell count is elevated in response to the infection.

? The low hemoglobin level is consistent with sickle cell anemia.

? Since she is homozygous (both genes coding for sickle hemoglobin), both

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her parents have sickle cell trait (heterozygous) and thus do not have

symptoms.

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? The diagnosis can be established with hemoglobin electrophoresis.

? Treatment includes searching for an underlying cause of crisis (infection,

hypoxia, fever, excessive exercise, and extreme changes in temperature),

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administration of oxygen, intravenous fluids for hydration, pain

management, and consideration of a blood transfusion.

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Proteome:

complete set of proteins expressed in a cell

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Genetic code:

A dictionary that identifies the correspondence

between a sequence of nucleotide bases and

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a sequence of amino acids.

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Features of the Genetic Code

? Degenerate

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? Unambiguous

? Nonoverlapping

? Not punctuated

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? Universal

In mitochondria: AUA= Met, UGA= trp;

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AGA&AGG= stop codon

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Recognition of the codon by the anticodon

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Involved in binding

tRNA to ribosome

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Serves as the

recognition site for enzyme:

adds amino acid

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Complimentary and antiparal el binding

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Adapted from Harper's Biochemistry
Point mutation: Single nucleotide change in coding region

1. Silent mutation

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2. Missense mutation

3. Nonsense mutation

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Adapted from Lippincott,s Biochemistry
Missense mutations resulting in abnormal hemoglobin chains

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Other mutations

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1. Trinucleotide repeat expansion

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Adapted from Lippincott,s Biochemistry

Fragile X

? Fragile X is the most common inherited form of

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mental retardation,

? Affecting primarily males

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? moderate to severe mental retardation,

hyperactivity, typical facies such as large jaw and

large ears. Pigmented skin lesions (cafe au lait)

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can also be seen.

? The fragile X mental retardation (FMR) gene

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product is affected and, through a little-

understood mechanism, leads to mental

retardation.

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Fragile X syndrome

? Molecular basis of disease:

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Mutation resulting in an increased number of

CGG repeats on the X chromosome. When the

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number of repeats reaches a critical size, it can be

methylated and inactivated resulting in the

disorder. Individuals who carry 50 to 199 repeats

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are phenotypical y normal and carry a

premutation. If repeats exceed 200, the patient

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has a full mutation; and if methylation occurs, he

or she wil be affected.

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? 2. Splice site mutation

? 3. Frameshift mutation

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? Deletion
? Addition

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Effects of deletions in a gene on the sequence of the mRNA

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transcript and of the polypeptide chain translated therefrom

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Adapted from Harper,s Biochemistry

The effects of insertions in a gene on the sequence of the mRNA

transcript and of the polypeptide chain translated therefrom

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The effects of deletions and insertions in a gene on the sequence of the mRNA

transcript and of the polypeptide chain translated there from

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Cystic fibrosis

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? is an inherited condition affecting approximately 1 in 2500

white individuals.

? Affected patients usually have abnormal mucus secretion

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and eccrine sweat glands leading to respiratory infections,

gastrointestinal obstruction, pancreatic enzyme dysfunction

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leading to malabsorption of nutrients, and excessive

electrolyte secretion.

? The protein cystic fibrosis transmembrane conductance

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regulator (CFTR) is defective, leading to abnormal chloride

transport.

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? Approximately 70 percent of mutations are accounted for

by deletion of three specific base pairs at the F 508 position

of the CFTR.

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Case 4

? A 40-year-old male returned from a deer-hunting trip approximately 6

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weeks ago, and presents to clinic with multiple complaints. He states that

recently he has had worsening joint pain and "arthritis" in multiple joints

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that seems to move to different spots. Patient also complains of some

numbness in his feet bilaterally.

? The patient denies any medical problems and he had a normal annual

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physical prior to hunting trip. On further questioning, he remembered

having a rash on his body and the lesions were circular and appeared to be

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resolving in the center. He noted that he felt really bad once he got home

with muscle ache (myalgias), joint ache (arthralgias), stiff neck, and severe

headache. He also remembered that many of his hunting friends had

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experienced flea and tick bites and is quite sure he was bitten as well. The

physical exam is essentially normal except some joint tenderness of left

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knee and right shoulder. After making your diagnosis you gave him a

prescription for erythromycin.

? What is the most likely diagnosis?

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? What is the biochemical mechanism of action of erythromycin?

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Steps of protein synthesis
Sequences of signal for initiation (Prokaryote and Eukaryote)

Peptide bond formation does not require energy? How it is possible

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DIFFERENCES IN PROKARYOTIC AND

EUKARYOTIC PROTEIN SYNTHESIS

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PROKARYOTE

EUKARYOTE

Start

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fMet-tRNA

Met-tRNA

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Recognition

Shine-Dalgarno

5 caps direct e-IFs

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sequence

sequence

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Initiation factors

IF-1, IF-2, IF-3

multiple e-Ifs (>10)

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Elongation factors EF-Tu, EF-G, EF-Ts

Multi-subunit

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eEF-1, eEF-2, eEF-3

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Components required for translation

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? Amino acids

? tRNA

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? Aminoacid attachment site

? Anticodon

? DHU loop

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? Pseudouridine loop

? Codon recognition by tRNA

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? Antiparal el binding between codon and anticodon

? Wobble hypothesis

? Aminoacyl tRNA synthetase

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Activation of Amino acid

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? Messenger RNA

? Functional y competent ribosome

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? rRNA

? Ribosomal protein

? A,P and E sites

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? Cel ular location

? Free in the cytosol

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? Associated with endoplasmic reticulum

? Protein factors for initiation, elongation and

termination

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? Energy sources

? Four high energy bonds cleavage for addition of

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one amino acid to the polypeptide chain

? 2 from ATP- aminoacyl tRNA synthetase reaction

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? 2 from GTP

? 1 for binding aminoacyl tRNA to the A site

? 1 for translocation

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? AT P and GTP molecules for initiation in eukaryotes

? GTP required for termination in both pro and Eu

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Steps of protein synthesis

? Initiation

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? Elongation
? Termination

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Adapted from Lippincott,s Biochemistry

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Adapted from Lippincott,s Biochemistry
Video on Prok translation

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Eukaryotic translation

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? Initiation:

? (1) dissociation of the ribosome into its 40S and 60S

subunits;

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? (2) binding of a ternary complex consisting of the

initiator methionyl-tRNA, (met-tRNAi), GTP, and eIF-2

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to the 40S ribosome to form the 43S preinitiation

complex;

? (3) binding of mRNA to the 40S preinitiation complex

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to form the 48S initiation complex; and

? (4) combination of the 48S initiation complex with the

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60S ribosomal subunit to form the 80S initiation

complex.

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Adapted from Harper,s Biochemistry

The circularization of mRNA through protein-protein interactions

between 7meG cap-bound elF4F and poly A tail-bound poly A binding protein

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Adapted from Harper,s Biochemistry
Activation of eIF-4E by insulin and formation

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of the cap binding eIF-4F complex

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Adapted from Harper,s Biochemistry

Elongation

? (1) binding of aminoacyl-tRNA to the A site,

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? (2) peptide bond formation,
? (3) translocation of the ribosome on the

mRNA, and

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? (4) expulsion of the deacylated tRNA from the

P- and E-sites.

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Diagrammatic representation of the peptide elongation

process of protein synthesis

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Adapted from Harper,s Biochemistry

Peptide elongation process of protein synthesis

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Adapted from Harper,s Biochemistry
Peptide elongation process of protein synthesis

Adapted from Harper,s Biochemistry

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Diagrammatic representation of the

termination process of protein synthesis

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Adapted from Harper,s Biochemistry
The termination process of protein synthesis

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Adapted from Harper,s Biochemistry

The termination process of protein synthesis

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Adapted from Harper,s Biochemistry
Comparison between Prokaryotes and Eukaryotes

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Cel

Factor

Function

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Initiation

P

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IF2 GTP

Bring charged initiating tRNA to P

E

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eIF2-GTP

site

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P

IF3

Prevent association of subunits

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E

eIF3

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Elongation

P

EF-Tu-GTP

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Bring al other charged tRNA to A site

E

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EF-1-GTP

P

EF-Ts

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Guanine nucleotide exchange factor

E

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EF-1

P

EF-G-GTP

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Translocation

E

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EF-2-GTP

Termination

P

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RF-1,2

Recognize stop codon

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E

eRF

P

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RF-3-GTP

Release of other RF

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E

eRF-3-GTP

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Video on EUK translation

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Clinical implication

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Adapted from Lippincott,s Biochemistry
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Adapted from Lippincott,s Biochemistry

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Post translational modification

A. Trimming

B. Covalent attachment

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Methylation

Acetylation

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Glycosylation

N glycosidic linkage

O glycosidic linkage

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Lipidation

GPI (glycosyl phosphatidyl inositol)

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S palmitoylation

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Post translational modification

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Phosphorylation

Formation of disulphide bonds

Gammacarboxylation----glutamic residue

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Hydroxylation ----Proline and lysine

C. Protein degradation

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Ubiquitination

Proteolysis

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MCQ1

? The 6-year-old son of a migrant worker is brought to a clinic with

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chil s, headache, nausea, vomiting, and sore throat. The examining

physician notes a persistent grayish colored membrane near the

tonsils. History reveals that the patient has not been immunized

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against diphtheria. Diphtheria toxin is potential y lethal in this

unimmunized patient because it causes which of the following?

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? A. Inactivates an elongation factor required for translocation in

protein synthesis

? B. Binds to the ribosome and prevents peptide bond formation

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? C. Prevents binding of mRNA to the 60S ribosomal subunit

? D. Inactivates an initiation factor

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? E. Inhibits the synthesis of aminoacyl-charged tRNA

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MCQ2

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? Many antimicrobials inhibit protein translation. Which

of the following antimicrobials is correctly paired with

its mechanism of action?

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? A. Tetracyclines inhibit peptidyltransferase.

? B. Diphtheria toxin binds to the 30S ribosomal subunit.

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? C. Puromycin inactivates EF-2.

? D. Clindamycin binds to the 30S ribosomal subunit.

? E. Erythromycin binds to the 50S ribosomal subunit.

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MCQ3

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? Translation of a synthetic polyribonucleotide containing the

repeating sequence CAA in a cell-free protein- synthesizing

system produces three homopolypeptides: polyglutamine,

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polyasparagine, and polythreonine. If the codon for

glutamine and asparagine are CAA and AAC, respectively,

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which of the following triplets is the codon for threonine?

? A. AAC.

? B. CAA.

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? C. CAC.

? D. CCA.

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? E. ACA.

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MCQ4

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? A tRNA molecule that is supposed to carry cysteine (tRNAcys) is

ischarged, so that it actual y carries alanine (ala-tRNAcys). Assuming

no correction occurs, what wil be the fate of this alanine residue

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during protein synthesis?

? A. It wil be incorporated into a protein in response to an alanine

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codon.

? B. It wil remain attached to the tRNA, as it cannot be

used for protein synthesis.

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? C. It wil be incorporated randomly at any codon.

? D. It wil be chemical y converted to cysteine by cel ular enzymes.

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? E. It wil be incorporated into a protein in response to

a

cysteine codon

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