Final Exam Information

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Wed, Dec 10 8:00 10:30 am rm 119

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Mon, Dec 8 11:00 am 1:30 pm rm 119

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CHAPTER 17

12-3-08

From Gene to Protein- Translation

Translation- key players

mRNA into protein rRNA- ribosomal RNAmakes up ribosomes

Ribosome-

Amino acids Polypeptide chain (protein)

Ribosome

couple mRNA
tRNA

with tRNA

tRNA- transfer RNA- carry (transfers) amino acids to ribosome

mRNA

tRNA

Transfers amino acids from cytoplasm to ribosome One tRNA for each amino acid Cell keeps cytoplasm stocked with tRNAs bound to amino acids

tRNA

Each tRNA carries a specific amino acid on one end Anticodon on the other end- base-pairs with a complementary codon on mRNA
Amino acid attachment site

tRNAs are recycled

Anticodon

Structure & Function of tRNA

Single RNA strand ~80 nucleotides long

3
Amino acid attachment site Amino acid attachment site
3

A C C

2D structure
3 Anticodon 5 Anticodon

Anticodon

3D structure

Amino acid

tRNA binds a specific amino acid

Catalyzed by aminoacyl synthetases 20 different synthetases- on for each amino acid
Pyrophosphate Phosphates tRNA

Aminoacyl-tRNA synthetase (enzyme)

Hydrolysis of ATP drives rxn

AMP

Aminoacyl tRNA (charge tRNA)

tRNA Binds mRNA

Correct match between tRNA anticodon & mRNA codon Only 45 tRNAs (not 61 as we would expect, as there are 61 different codons)

Some tRNAs can bind more than 1 codon Wobble room

Ribosomes

Facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis Two ribosomal subunits (large and small)- made of proteins & ribosomal RNA (rRNA)
Large subunit

Tetracycline & streptomycininhibit the ability of prokaryote ribosomes

Small subunit

Ribosome Binding Sites

Holds the tRNA that carries the growing polypeptide chain Holds the tRNA that carries the next amino acid to be added to the chain

P site
(Peptidyl-tRNA binding site)

A site
(Aminoacyl-tRNA binding site)

E site
(Exit site)

Where discharged tRNAs leave the ribosome

mRNA binding site

Large subunit

Small subunit

Ribosome holds tRNA & mRNA in close proximity

Amino end
Growing polypeptide

Next amino acid to be added to polypeptide chain

E mRNA tRNA 3

Codons

Ribosome catalyzes formation of peptide bond between amino acids

Translation- Building a Polypeptide

The three stages of translation:

Initiation
Elongation Termination

All three stages require protein factors that aid in the translation process

Translation- Initiation Stage

Large ribosomal subunit

Establishes reading frame

Initiator tRNA mRNA

P site

Requires energy
GTP GDP E

A
3

5
Start codon mRNA binding site

3
Small ribosomal subunit

Translation initiation complex

Small ribosomal subunit binds with mRNA & a special initiator tRNA, then moves along the mRNA until it reaches the start codon (AUG)

Proteins called initiation factors bring in the large subunit, the initiator tRNA occupies the P site

Elongation of the Polypeptide Chain

Amino acids added one by one to the preceding amino acid Each addition involves proteins called elongation factors Occurs in 3 steps:
Codon

recognition Peptide bond formation Translocation

Amino end of polypeptide

1. Codon Recognition
Anticodon of incoming tRNA basepairs with complementary mRNA codon in A site
Ribosome moves along mRNA 5 to 3 direction

E mRNA

3 P A site site 2 GTP 2 GDP

Ribosome ready for next aminoacyl tRNA

Hydrolysis of GTP inc efficiency and accuracy

E

3. Translocation Ribosome translocates the tRNA in A site to the P site, at same time empty tRNA in P site moves to E sitereleases

GDP GTP

2. Peptide Bond Formation

rRNA of large subunit catalyzes formation of peptide bond between new AA & carboxyl end of the growing chain in the P site

Polypeptide chain is synthesized in one direction

From Nterminus (amino end) to the C-terminus (carboxyl end)

TERMINATION OF TRANSLATION
Release factor
Free polypeptide
5 3 3 3

5
Stop codon (UAG, UAA, or UGA)

Ribosome reaches a stop codon on mRNA, the A site accepts release factor instead of tRNA.

Release factor hydrolyzes the bond between tRNA in the P site & the last AA of the polypeptide chain. Polypeptide Is freed from the ribosome.

Two ribosomal subunits & the other components of the assembly dissociate.

Polyribosomes

Normally, multiple ribosomes translate mRNA at the same time Use 1 mRNA transcript to make lots of protein Polyribosomes enable a cell to make many copies of a polypeptide very quickly
Growing polypeptides Incoming ribosomal subunits Completed polypeptides

Start of mRNA (5 end)

End of mRNA (3 end)

Completing the Functional Protein

Translation- not sufficient to make a functional protein

Needs

to be modified, folded

Completed proteins are targeted to specific sites in the cell

Protein Folding Modifications

During & after synthesis, polypeptide chain spontaneously coils & folds into its 3D shape
Amino

acid interactions

Gene determines 1 structure, 1 structure in turn determines shape Chaperone proteins- help fold protein correctly

Post-Translational Modifications

Certain amino acids chemically modified by attachment of sugars, lipids, phosphate groups Enzymes may remove 1 or more amino acids from the leading (amino) end of the chain Polypeptide chain may be enzymatically cleaved into 2 or more pieces- insulin 2 or more polypeptide chains come together to form subunits of one protein- hemoglobin

Targeting Polypeptides to Specific Locations

Free ribsomes (cytosol)- synthesize proteins that function in the cytosol Bound ribosomes (attached to ER)- make proteins of the endomembrane system & secreted proteins Ribosomes are identical & can switch from free to bound

Targeting Polypeptides to Specific Locations

Polypeptide synthesis always begins in the cytosol Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide Signal peptide recognized- signal-recognition particle (SRP) SRP brings the signal peptide & its ribosome to a receptor protein built into the ER membrane

Polypeptide synthesis begins on a free ribosome in cytosol

Ribosomes

SRP binds receptor protein in ER membrane

SRP binds signal peptide

SRP leaves, polypeptide synthesis resumes, simultaneous translocation across the membrane

enzyme cuts off signal peptide

Rest of the completed polypeptide leaves ribosome & folds into its final Signal cleaving conformation

mRNA Signal peptide Signalrecognition particle (SRP) Signal peptide removed

ER membrane

SRP receptor CYTOSOL protein

Protein

Membrane pore & signal cleaving enzyme

ER LUMEN

Translocation complex

RNA plays multiple roles in the cell

Type of RNA Messenger RNA (mRNA) Functions

Carries information specifying amino acid sequences of proteins from DNA to ribosomes Transfer RNA Serves as adapter molecule in (tRNA) protein synthesis; translates mRNA codons into amino acids Ribosomal Plays catalytic (ribozyme) roles RNA (rRNA) and structural roles in ribosomes

TRANSCRIPTION

DNA 3

5 RNA transcript RNA PROCESSING

RNA polymerase Exon RNA transcript (pre-mRNA) Intron

Aminoacyl-tRNA synthetase
NUCLEUS FORMATION OF INITIATION COMPLEX CYTOPLASM mRNA AA tRNA AMINO ACID ACTIVATION

Growing polypeptide

A P E Ribosomal subunits

Activated amino acid

5 E A

TRANSLATION Anticodon

Codon Ribosome

Point mutations

Point mutations- chemical changes in just one base pair of a gene Point mutations divided into 2 general categories:
Base-pair
Silent Missense

substitutions

Nonsense

Base-pair

insertions or deletions

Substitutions

Replaces one nucleotide and its partner with another pair of nucleotides Silent mutation- results in same amino acid Missense mutations- code for an different amino acid Nonsense mutations- change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Base pair substitution- missense mutation

Wild-type hemoglobin DNA
3 5 3

Mutant hemoglobin DNA

5

mRNA

mRNA

Normal hemoglobin

Sickle-cell hemoglobin

Sickle Cell Anemia

Wild type mRNA 5 Protein Amino end Base-pair substitution Stop Carboxyl end 3

Silent
U instead of C

Stop

Missense
A instead of G

Stop

Nonsense
U instead of A

Stop

Insertions and Deletions

Insertion- addition & deletions are losses of nucleotide pairs in a gene Much more severe effects on resulting protein than substitutions do May alter the reading frame, producing a frameshift mutation

Wild type

mRNA 5 Protein
Amino end Base-pair insertion or deletion

3 Stop Carboxyl end

Frameshift causing immediate nonsense

Extra U

Stop

Frameshift causing extensive missense

Missing

Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid

Missing

Stop

Mutagens

Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens- physical or chemical agents that can cause mutations
X-rays
UV

light Base analogs- resemble DNA bases