L8 - Gene To Protein Flashcards
Genotype
Organism’s hereditary info
Phenotype
Actual observable/physiological traits
= genotype + interaction w/environ.
Control points of gene expression
- transcription
- processing of pre-mRNA
- transport
- translation
Importance of control points
Gene expression tightly regulated to achieve right thing at right time in the right place (temporal/spatial control)
DNA orientation
Upstream = promotor end = 3’ end of template strand = 5’ end of non-template strand
- has control elements that dictate things need to be assembled: when and how
Downstream = terminator end = 5’ end of template strand = 3’ end of non-template strand
Purines
Adenine, guanine
Pyrimidine
Thymine, cytosine
Transcription initiation
1) eukaryotic promotor (TATA box sequence ~25 nt upstream)
2) promotor assemble transcription factors (including TATA box binding protein, TBP) - allows RNA polymerase II to bind
3) transcription factors + RNA polymerase II (teardrop shaped enzyme) form transcription initiation complex
Transcription elongation
10-20 nucleotides exposed at a time when DNA unwound (H-bonds broken)
- H-bonds between RNA nucleotides and DNA bases
- phosphodiester bonds between RNA nucleotides
Transcription termination
1) polyadenylation signal (AAUAAAA)
2) pre-mRNA released, RNA polymerase dissociated
Fidelity (proofreading)
Less than for DNA replication
UTR
Untranslated regions at 5’ and 3’ ends
Steps of processing pre-mRNA transcript
Capping, tailing, splicing
Capping
Modified guanine added to 5’ end
Tailing
50-250 adenine nucleotides (polyA) added (strand is polyadenylated) to 3’ end to form poly-A-tail
Splicing location
Spliceosome
- large complex of proteins and small RNA
- in nucleus
Alternative splicing
- process by which different combos of exons are joined together
- results in production of multiple forms of mRNA from single pre-mRNA
- 1 gene to multiple gene products
Translation components
Ribosomes, tRNA
Ribosome sites
- mRNA binding site on small subunit
- tRNA binding sites on large subunit
tRNA binding sites
- A (aminoacyl-tRNA binding) site: holds next-in-line tRNA
- P (peptides-tRNA binding) site: holds tRNA carrying the growing polypeptide
- E (exit) site: tRNAs exit from here
Translation process
- codons translated into amino acids
- protein synthesis catalysed by ribosomes
Initiator tRNA
TRNA carrying methionine (Met)
Translation initiation
1) small ribosomal subunit with initiator tRNA + bind to 5’ cap end of mRNA
2) small ribosomal subunit scans downstream to find translation start site (AUG)
3) H-bond between anticodon/mRNA
4) large ribosomal subunit binds = initiation complex
GTP required
GTP
Guanosine triphosphate
Translation elongation
1) codon recognition
- tRNA in cytosol + complementary codon
- GTP invested to increase accuracy/efficiency
2) peptide bond formation
- large subunit rRNA catalyses peptide bond
- polypeptide moves from P site tRNA to A site tRNA
3) translocation
- P site tRNA moves to E site then released
- A site tRNA moves to P site
- GTP required
Empty tRNA reloading
In cytoplasm using aminoacyl-tRNA synthetases (family of related enzymes e.g tyrosyl-tRNA synthetase)
Amino acid-tRNA bond
Covalent
Translation termination
1) stop codon
2) release factor to A site
3) release factor promotes hydrolysis of bond between P site tRNA and last amino acid
4) polypeptide released
5) ribosomal subunits + other components dissociate and recycled
- hydrolysis of TWO GTP molecules required
Nascent
Growing (e.g polypeptide)
Polypeptide orientation
N terminus (amino end) = 5’ end of mRNA C-terminus (carboxyl end) = 3’ end of mRNA
R group of amino acid
Side chains determine property of each amino acid and therefore final structure and function of protein
- nonpolar = hydrophobic
- polar= hydrophilic
- electrically charged = hydrophilic (acidic-negatively charged, basic-positively charged)
No. Amino acids
Twenty standard (coded for) amino acids
Protein structures
Primary, secondary, tertiary, quarternary
- all proteins go to tertiary
Primary structure
- determined by sequence
- held by covalent bonds
- starts to form secondary structures as soon as it leaves ribosome (Protein assumes 3D conformation either spontaneously as it emerges from ribosome or with help of chaperones)
Chaperones
Proteins that assist with protein folding
one type provides the right conditions for folding e.g pH, conc. etc.
Secondary structure
- can be predicted by sequence
- held by weak H-bonds
- alpha helix or beta pleated sheets
Tertiary structure
- less predictable
- 3D shape stabilised by side chain interactions
Quarternary structure
Multiple proteins associate together to form functional protein
E.g hemoglobin
Functional classification of proteins
Housekeeping proteins, other proteins
Housekeeping proteins
continuously produced
- commonly used
- protein/mRNA present in large quantities
- (typically) longer half-life in cells
Other proteins
Produced in response to stimuli as required
- cell signalling
- signal transducted and many enter nucleus to activate transcription
- results in production of short-lived protein to carry out required function
Translation location
All commence on free ribosomes
1) cytosolic proteins - complete on free ribosomes
2) proteins going through endomembrane system (RER and Golgi) - complete translation at fixed ribosomes on RER (target specific receptors)
Signal peptides
Located at N terminus of protein (~20 amino acids) and are what direct ribosomes to RER
SRP
Signal recognition particle
Signalling process
1) polypeptide synthesis begins
2) SRP binds to signal peptide which pauses translation
3) SRP binds to receptor protein on RER
4) SRP detaches and polypeptide synthesis resumes
5) signal sequence inserts into ER membrane and nascent chain is translocated across
6) signal peptidase (signal-cleaving enzyme) cuts off signal peptide
7) completed polypeptide folds into final conformation
Secretory proteins
Solubilised in ER lumen
E.g insulin
Membrane protein
- Remains anchored to membrane (one or more hydrophobic segments of polypeptide anchor it in the bilayer)
- vesicle forms around it
Vesicles go to
Golgi for further maturation then to plasma membrane where they are
- secreted
- remain at cell surface
- targeted to lysosomes where they serve degradation of nutrients and facilitate renewal of the cell’s own macromolecules
Post-translational modifications
Confer activity - phosphorylation - cleavage Ability to interact with other molecules - methylation - biotinylation Direct to particular locations - ubiquitination Other - acetylation - carboxylation - carbohydrate addition
Post-translation modification location
- some in golgi
- others in cytosol
Gene examples
- TATA-box binding protein (TBP)
- Huntington (HTT)
Genome
All DNA as a whole
~3000 Mbp
~20,000 genes
if translation terminated at wrong place for cytosolic/proteins going through endomembrane system
proteins won’t fold correctly (wrong conditions) = non-functional protein
secretory and membrane proteins terminate translation
on rough ER
sorting error
protein correct but in wrong place
- may have fully folded and modified protein but trafficked incorrectly so can’t do its job
failure of post-translational modifications
non functional protein
poor proofreading
transcript present but may or may not function as a protein depending on effect on amino acid
failure of splicing
introns not removed = have a pre-mRNA but correct protein won’t be made
absence of RNApol II or other required factor
no transcription = no transcript at all
incorrect folding of protein
non-functional protein
effect of errors
if only a small proportion of products affected, may not be catastrophic
