Theme 2C Flashcards
Protein Structure & Translational and Posttranslational Gene Regulation
What are the three steps of translational regulation of gene expression?
- Initiation
- Elongation
- Termination
What is Translation?
assembly of amino acids into polypeptides
aa contain amino & carboxyl group bonded to central carbon (a) with a hydrogen and R group
Two amino acids are joined together by a covalent ________ bond between the ______ and ________ by a ____________
peptide; amino; carboxyl; dehydration reaction
Polypwptides are
linear chains of amino acids linked by peptide bonds
Type of Amino Acid
Non-polar Amino Acid
R groups usually contain -CH2 or -CH3
Type of Amino Acid
Uncharged polar amino acid
R groups usually contain oxygen (-OH)
Type of Amino Acid
Charged amino acid
R groups that contain acids or bases that can ionize
Type of Amino Acid
Aromatic amino acid
R groups contain a carbon ring with alternating single and double bonds
Special Functional amino acid
Methionine
first amino acid in polypeptide
Special Functional amino acid
Proline
causes kink in polypeptide chains
Special Functional amino acid
Cysteine (S-S)
What does the S-S contribute to?
disulfide bridge contributes to structure of polypeptides
Levels of protein structure
Primary amino acid sequence
what does it determine?
protein folding and 3-D structure which is critical for proper function
Levels of protein structure
2º Structure
what does it depend on?
hydrogen bonding in polypeptide backbone (a-helices & b-sheets)
Levels of protein structure
3 structure
3-D structure of a single polypeptide and is composed of interactions between amino acid side chains
Levels of protein structure
4 structure
are interactions between more than one polypeptide to form a multisubunit protein
(ie. hemoglobin)
Levels of protein structure
Protein folding is disrupted by ______ or ________
denaturation or mutations that change amino acid sequence
Levels of protein structure
Chaperones function to
protect slow-folding or denatured proteins by preventing their aggregation
tRNAs
adaptors between codons (mRNA) and amino acids
slide 6 - tRNA
2-D cloverleaf and 3-D L-shaped folded RNA molecule from
self complementarity
tRNA
Acceptor stem is
where the amino acid is attached and contains the sequence 5’-CCA-3’ at the 3’ end of the tRNA
tRNA
The anti-codon is the ________ loop of the cloverleaf and contains three nucleotide sequence that recognize the ___________ by _______________ with mRNA
bottom; codon; base pairing
What type of base pairing?
____________ base pairing between codon and anticodon in tRNA
antiparallel
Aminoacyl-tRNA charging
Aminoacyl-tRNA synthease adds
(charging) adding the amino acid to the tRNA
the amino acid to the acceptor stem of the correct tRNA
Aminoacyl-tRNA (Charging)
Charging Reaction (Aminoacylation)
What is the equation?
amino acid + tRNA + ATP –> aminoacyl-tRNA + AMP + PPi
The genetic code consists of
2 things
61 “sense” codons and amino acids specified by them
Genetic code
The codons are written
in what direction?
5’-3’, as they appear on mRNA
Genetic Code
AUG (Methionine)
What is it?
initiation (start) codon
Genetic code
UAA, UAG, UGA
are what type of codons?
termination (stop) codons
Genetic Code
The genetic code shows _________ in which an amino acid can be specified by _____________
degeneracy; more than one codon
Rules of the Genetic Code
In what direction are codons on mRNA read?
5’-3’ direction
Rules of Genetic Code
Codons are ______________________ and the message contains no __________
non-overlapping; gaps
Rules of Genetic Code
The message is translated in a ________________ by the start codon
fixed reading frame
Frames
Is it neccessary that translation always start at the start codon?
no
What are the three reading frames
1+, 2+, 3+
Why are there only three and not four frames?
Because 4+ would be the same as 1+
What is the most likely frame that encodes for polypeptide
2+
Are there 61 different tRNAs that bind to the 61 mRNA codons?
no
There are less than 61 tRNAs because
tRNAs can read more than one codon
Why can tRNAs read more than one codon?
Because base at 5’ end of anticodon can form H-bonds with more than one type of base located at the 3’ end of a codon “wobble”
Is there wobble during pairing of the other two nucleotides in the anti codon with the codon
not the nucleotide at 5’ end
No
Ribosome
(prokaryotes)
protein synthesis machinery
Ribosome is composed of ___________________ made of a complex of ______________ and ________________
two subunits; proteins; rRNA
Ribosomes (prokaryotes)
The large subunit (50S) is made of ________________ and contains _________________________ for _________________.
5S and 23S rRNA + 34 proteins; peptidyltransferase center; formation of peptide bonds
Ribosomes (prokaryotes)
Small subunit (30S) is made of ________________ and contains ____________ where ____________________ & ________________.
16S rRNA + 21 proteins; decoding center; charged tRNAs read and decode the codon of mRNA
based on anti-codon codon binding through parallel complimentary base-pairing
Ribosomes (prokaryotes)
S (svedberg unit)
measure of seidmentation velocity, and, therefore, mass
Ribosomes (prokaryotes)
each subunit exists separately in the ___________ but the two join together on the __________ molecule
cytoplasm; mRNA
tRNA binding sites of ribosomes
What are the three tRNA binding sites of ribosomes?
- Aminoacyl (A)
- Peptidyl (P)
- Exit (E)
tRNA binding sites of ribosomes
P site (peptidyl)
binds to the tRNA attached to the growing peptide chain
tRNA binding sites of ribosomes
A site (aminoacyl)
binds to the tRNA carrying the next amino acid to be added
tRNA binding sites of ribosomes
E site (exit)
binds to the tRNA that carried the previous amino acid added
Translation Initiation (eukaryotes)
An ______________________ containing the ribosome, mRNA, and the initiator tRNA bound to methionine is formed during translation initiation
initiation complex
What are the steps for translation initiation in eukaryotes
- initiator tRNA met is brought to the P-site of the small ribosome subunit (reaction requires GTP)
- complex of initiator tRNAmet and small ribosomal subunit is recruited to the capped 5’-end of mRNA & scans along the mRNA in a 5’-3’ direction until it reaches the first 5’-AUG-3’ start codon
- complimentary base pairing occurs between anticodon of initiator tRNAmet and start codon of mRNA
- Large ribosomalsubunit binds to small subunit to form the initiation complex, which is now ready to accept the first tRNA in the A-site
- GTP is hydrolyzed to GDP and translation begins
Translation elongation: growth of the polypeptide
What are the steps?
4 steps
- correct aminoacyl tRNA is loaded to A site by Elongation Factor-GTP (tRNA anticodon is compliemntary to mRNA codon in the A-site)
- Peptidyl transferase in the large subunit forms a peptide bond between carboxyl group of growing polypeptide and the amino group of the amino acid in the A site (tRNA in P site is uncharged)
- Ribosome translocate in which the tRNA with the polypeptide shifts from A to P site, and uncharged tRNA shifts from P to E site where it is ejected
- The next codon of the mRNA is now A site and next aminoacyl tRNA can be loaded by EF-GTP
The three stop codons are found at _________________
end of a protein coding sequence
UAA, UAG, UGA
Stop codons are recognized by
proteins called release factor
when ribosome reaches the stop codon, the release factor binds the A-site and stimulates __________________________________________ to _______________________________
peptidyl transferase; cleave polypeptide from P-site tRNA
Part 2
Posttranslational Regulation of Gene Expression
3 STEPS
- Phosphorylation
- Ubiquitination
- Proteolysis
Posttranslational regulation of proteins
Phosphorylation
addition of phosphate to proteins by kinases, which can activate or inhibit their activity
Example: CDK1 phosphorylates and activates MAP to promote spindle assembly and mitotic entry
Posttranslational regulation of proteins
Ubiquitination
addition of ubiquitin molecules to proteins target them for destruction by the proteasome
Example: cylclin is destroyed at the end of mitosis to inactivate CDK1
Posttranslational regulation of proteins
Proteolysis
specific cleavage of the protein can induce activity
done by enzyme protease
Example: proteolysis of viral envelope glycoprotein triggers maturation of HIV
Epigenetics
posttranslational modification of histones that affect transcription
Epigenetics
Changes in gene transcription occurs ______________________
without changes in DNA sequence
Epigenetics
Modifications to ______________ on ______________________ affect transcription of genes (histone code). This is a translational/posttranslational event.
lysines; histone tails (have + charge); posttranslational
Epigenetics
Histone acetyltransferase (HATs) that add _________________ to histone tails increase gene transcription by losening DNA binding! Why?
acetyl groups (CH3CO-); because histones negative and DNA is negative, so repulsive forces causes DNA to loosen
Epigenetics
___________________ of histone tails can activate and repress transcription of genes
methylation
Epigenetics
DNA can be _________________________ at ________________ close to promoter repressing transcription
methylated; CpG islands
Epigenetics
______________________________ displaces nucleosomes from promoter regions (ATP-dependent) activating transcription
Chromatin remodelling complex
Level of gene regulation
Translational regulation
control of protein synthesis (rate of trnaslation initiation or formation of the initiation complex)
Level of gene regulation
Posttranslational regulation
control of protein abundance & activity
ie. availability of functional proteins
Level of gene regulation
Abundance of protein depends on its rate of _________________________ (translation initiation) and ____________________ (posttranslational)
synthesis; degradation
Level of gene regulation
Activity of protein depends on ___________________________ and _____________________________ (cleavage)
posttranslational modifications; processing
Level of gene regulation
The expression level of a specific gene depends on the ______________________ & ___________________
abundance of proteins; its activity
rEVIEW
slide 20
4 Levels
Levels of Eukaryotic Gene Regulation
- Epigenetics (Chromatin remodelling)
- Transcriptional (mRNA synthesis)
- Post-transcriptional (mRNA processing)
- Translation (protein synthesis)
- Post-translation (protein processing)
Levels of Eukaryotic Gene Regulation
Changes at any level of gene regulation would potentially _______________
alter protein expression and cause variation in phenotype
