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Exam 3 Material: Translation
Exam 3 Material: Translation
27
Biochemistry
Graduate
11/11/2014

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Term
Translation Basics
Definition
Translation is the production of a protein through the use of mRNA as the carrier of information.
It is carried out by the ribosome, a huge complex of proteins and integral RNA molecules (ribosomal RNA, or rRNA)
The ribosome uses activated amino acids as the substrate for polymerization
Amino acids are activated when they combine with transfer RNA (tRNA) for use in the ribosome
Term
Codons
Definition
The mRNA is read as blocks of three sequential nucleotides (codon), which specifies a particular amino acid
-Codons do not overlap, no gaps
-Three possible reading frames, could start at any base
-Correct reading frame is specified by an initiation codon, generally AUG, as well as regulatory sites for correct positioning
-Wrong reading frame = wrong proteins
Term
tRNA*
Definition
True translational molecule, having an activated amino acid on one side, and the matching anticodon on the other.
- 3 stem-loop patterns
- Altered bases created in nucleus
- Side loops have important functions
Term
D loop of tRNA
Definition
needed for synthetase recognition
Term
Aminoacyl-tRNA synthetase
Definition
attaches AMP to the a.a., then a.a. to the 2' or 3' hydroxyl of the terminal adenine residue. Each a.a. has its own synthetase
Term
Activation of Amino Acids
Definition
must be activated before they are polymerized into peptide chains. Activated by joining them to tRNA.
-First step is formation of a mixed anhydride link between the amino acid and AMP, giving an aminoacyl adenylate.
-Second step forms an aminoacyl-tRNA, a charged tRNA. This is the substrate for polymerization into a polypeptide chain.
Term
Ribosome Structure
Definition
Composed of several rRNA strands and many proteins.
-Ribosomal proteins are located mainly on the exterior surface of the ribosome, while RNA occupies the interior.
-Ribosomal RNA is folded into domains that contain a few hundred nucleotides. Domains are composed of helices, many terminating in hairpin turns, and with bulges or loops of unequal numbers of bases which introduce bends into the helix
- Many of the nucleotides are modified
Term
Ribosome Size(s)
Definition
Bacterial ribosome 70s
- 30S and 50s subunits
Eukaryotic ribosome: 80s
-60s and 40s subunits
Term
Snourps
Definition
help to modify rRNA
Term
Comparing Prokaryotic and Eukaryotic Translation
Definition
Start codon: typically AUG
Prokaryotes use shine-dalgarno sequence to specify the position of the initiating codon.
Eukaryotes use the AUG codon nearest the 5' end of the mRNA
Bacteria and mitochondria start protein synthesis with a formylated methionine residue (fMEt), carried by a special tRNA
In protein synthesis at the endoplasmic reticulum in eukaryotes, the first amino acid incorporated into a nascent polypeptide chain willy typically aslo be methionine (but not fMet), which also is carried by a special intiating tRNA
Term
Polypetide Synthesis
Definition
Prokaryotes: Occurs in the cytosol
Eucaryotes: takes place mainly on the outer face of the membranes of the ER
-small amount takes place from free ribosomes int he cytoplasm
Three main stages to protein synthesis: intiation, elongation, termination.
Term
tRNA Binding and Chain Initiation
Definition
Ribosome has large and small subunit.
3 Binding sites:
1) Aminoacyl (A)
2) Peptidyl (P)
3) Exit (3) for uncharged tRNA
When two charged tRNA occupy A and P sites, a peptide bond will be formed.
Nascent peptide chain exits through the bottom of the large subunit.
- In eukaryotes, usually an exit to the ER lumen
Term
Sequence of Events
Definition
mRNA binds small subunit
-start codon of mRNA is positioned at the P site
-Initial charged tRNA binds to the start codon on the mRNA (usually methionine, fMet for bacteria)
-Large ribosomal subunit associates with the small subunit
+ This process is aided in Eu by proteins called initiation factors, and require GTP hydrolysis
- T loop of tRNA molecules is used to recognize the loading site on the ribosome
Term
T Loop of RNA
Definition
used to recognize loading site on the ribosome
Term
Elongation
Definition
Ribosome moves along mRNA sequentially by codon, matching each with an anticodon.
-At each step, the discharged tRNA exits through the E site, and the next tRNA enters the A site.
-Peptide bond forms between the nascent peptide chain and the next amino acid
-Elongation factors and GTP hydrolysis is required
-Peptide chain growns in the amino-to-carboxy direction, as the ribosome reads the mRNA in 5'to 3' direction
Term
Elongation factors
Definition
EF-TU and EF-G in bacteria
EF1 and EF2 in eukaryotes
provide speed and accuracy
Term
Termination Phase
Definition
Elongation continues until finally a termination (stop) codon on the mRNA is reached.
-No tRNA added to the A site
-Instead, a release factor fills the A site
-The ribosome dissociates into small an dlarge subunits, and the mRNA and tRNA molecules dissociate from the ribosome.
-The protein is folded into its tertiary state, often with the help of chaperones
Term
Characteristics of Eukaryotic Translation
Definition
Most ribosomes will dock at receptor on ER; the ribosome starts translation but pauses before docking.
-Signal recognition particle (SRP) attaches to the protein to help it find the ER site.
-Proteins that will be associated with a membrane will contain a special sequence to direct this process
-In the ER lumen, the protein folds and is modified. Transport vesicles then bud off the ER and carry proteins to the golgi for further processing, or to other compartments in the cell, or to the plasma membrane.
-Eukaryotes use signaling sequences to direct proteins to different cellular locations
Term
Selenocysteine*
Definition
Encoded by UGA (normally a stop codon)
A SECIS element (hairpin) in the mRNA allows its incorporation.
High reduction potential, found in enzymes involved in anti-oxidation
About 25 known human proteins with selenocysteine
Term
Ribosome Inhibitors
Definition
The ribosome is a big target for antibiotics.
-Bacterial and eukaryotic ribosomes differ in structure and sequence, and these differences can be targeted for antibiotic action against bacteria.
-Drugs known to inhibit Eu translation: puromycin, cycloheximide, anisomycin
Term
Tetracyclines
Definition
Interfere with decoding of mRNA by blocking tRNA from binding in the A site.
-Tetracycline, doxycycline, minocycline, tigecycline
-Broad spectrum
[Four rings with C2H6N)
Term
Macrolides
Definition
Bind around exit tunnel on the large ribosomal subunit, blocking peptide elongation
-Erythromycin,azithromycin (Zithromax), clarithromycin (Biaxin)

Broad Spectrum
Term
Aminoglycosides
Definition
Interfere with chain translocation; they can also interfere with proofreading - leading to errors in amino acid incorporation in the polypeptide, which in turn can lead to non-functional (nonsense) proteins that are toxic to the cell
-streptomycin,kanamycin,tobramycin,gentamicin,amikacin,spectinomycin

Broad spectrum
Term
Nitrofurans
Definition
Useful for UTIs caused by Gram (-) rods.

Weird pentose nitro things
Term
Chloramphenicol
Definition
Chloramphenicol interferes with peptide bond formation by blocking the A site.
Looks like a pikmin sitting down
-SIGNIFICANT TOXICITY = RARELY USED
Term
Reverse Transcriptase Inhibitors
Definition
Azidothymidine (AZT) is an analog of thymidine, and is an effective inhibitor of HIV replication.
-Taken up by the cella nd converted to AZT triphosphate, which is a substrate for HIV reverse transcriptase
- Like acyclovir, AZT lacks a 3' OH , so if it is incorporated at the end of a growing strand of DNA< there is no way to extend the DNA chain past it. This will block DNA synthesis and inhibit viral replication.
Term
Retroviruses and Reverse Transcriptases
Definition
Carry RNA, not DNA and often one or more copies of an RNA-dependent DNA polymerase, or reverse transcriptase. Enzyme syntesizes DNA, forming hybrid duplex, reverse transcriptase then degrades the RNA strand and syntesizes new DNA strand. Viral DNA may insert into host genome, then be transcribed.
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