Lecture 9: CSF Gene to Protein Flashcards
What is genotype?
An organism’s hereditary information
What is phenotype?
Actual observable or physiological traits
What is gene expression?
The process of forming a functional product from DNA
DNA -> RNA -> Protein
What are the 3 steps to gene expression?
Transcription
Processing
Translation
What are the 3 steps to transcription?
Initiation -> polymerase binds to promoter
Elongation -> moves downstream through the gene, transcribing RNA
Termination -> detaches after terminator reached
What is the difference in bases in mRNA and DNA?
Uracil takes place of thymine in mRNA
What is the significance of uracil replacing thymine in mRNA?
Thymine is more stable -> DNA is the inherited material hence needs to be more stable
Explain how initiation process occurs during transcription
Initiation is the assembly of proteins required before transcription can commence
- DNA contains a promoter region in which the TATA box can be found
- Several transcription factors bind to the promoter region (including TATA box binding protein [TBP])
- This changes the shape of the promoter region allowing RNA polymerase II to bind along with more transcription factors to form the transcription initiation complex
What is the TATA box?
A sequence of DNA found in the promoter region which marks where transcription should be initiated
What is TBP?
TATA box binding protein is a transcription factor that binds to the TATA box of the promoter region forming transcription initiation complex.
Explain how the elongation process occurs during transcription
10 - 20 nucleotides become exposed at a time as DNA unwinds
Complementary RNA nucleotides are added to the 3 to 5 DNA sequence (template strand).
As complementary base pairs form between mRNA and DNA, the 3’ end forms a phosphodiester bond with the 5’ end of incoming nucleotide.
Double helix reforms as transcript leaves the template strand
Explain how the termination process occurs for transcription
After transcription of the polyadenylation signal, nuclear enzymes release the pre-mRNA and RNA polymerase then dissociates from the DNA
What is the polyadenylation signal?
A sequence of nucleotide bases that signal the termination of transcription hence the release of pre-mRNA and RNA polymerase
What are the 3 steps to processing stage of gene expression?
Capping, tailing, splicing
What are exons?
Coding regions of mRNA strand including untranslated regions (UTR)
What are introns?
Non-coding regions intervening exons
What is UTR?
Untranslated regions at 5’ and 3’ ends of the mRNA
What is capping?
A modified guanine nucleotide is added to the 5’ end (lid on top)
What is tailing?
50-250 adenine nucleotides are added to the 3’ end (tail on bottom)
What is splicing?
Introns are removed from the transcript and adjacent exon ends are joined to form mature mRNA
Where does splicing occur?
At the spliceosome, in the nucleus
What is a spliceosome?
A large complex of proteins and small RNAs
What is alternative splicing?
A process in which different combinations of adjacent exons are joined together - this means multiple mRNA molecules can be formed from a single pre-mRNA
What is a codon?
A base triplet that codes for an amino acid
What are the 3 steps of translation?
Same as transcription -> initiation, elongation, termination
How are mRNA codons translated to tRNA molecules?
By attaching to ribosomes
Explain the structure of the ribosome
Large and small subunit
Large subunit contains E, P, and A sites (where tRNA binds to and exits)
Small subunit contains mRNA binding site
What is the A site?
A region on the large subunit of ribosomes where the next in line tRNA molecule is bound to
What is the P site?
A region on the large subunit of ribosomes where the tRNA carrying the growing polypeptide binds to
What is the E site?
A region on the large subunit of ribosomes where the tRNA that have added its amino acid to the polypeptide chain exit through
What is a tRNA molecule?
Transfer RNAs are the physical link between the amino acid and the mRNA.
Contains an amino acid attachment site and an anti-codon (3’ to 5’) complementary to the codon (5’ to 3’) on mRNA
How does the initiation stage of translation occur?
- Initiator tRNA (Met) is bound to the small subunit of the ribosome which binds to the 5’ cap of mRNA
- The small subunit ribosome scans the mRNA molecule in the downstream direction to find translation start site (AUG codon)
- At this start site hydrogen bonds form between the initiator anticodon (UAC) and mRNA (AUG)
- Large ribosomal subunit then binds completing the initiation complex (requires energy in the form of GTP)
How does the elongation stage of translation occur?
- Codon recognition - tRNA molecules with anticodon complementary to the codon of mRNA form hydrogen bonds using energy from GTP (these “next in line” tRNA will bind to the A site)
- A large subunit rRNA catalyses the peptide bond formation of adjacent amino acids of the P site and A site tRNA molecules -> the P site tRNA amino acid bond is broken (hence polypeptide chain only attached to A site tRNA)
- The tRNA from the A site moves into the P site and the tRNA in the P site exits through E site - again energy is required
- Emptied tRNA molecules with no amino acid attached are reloaded with the amino acid in the cytoplasm using aminoacyl-tRNA synthetases
What are aminoacyl-tRNA synthetases?
Enzymes which reload the tRNA molecules with no amino acid attached in the cytoplasm
How does the termination stage of translation occur?
- The ribosome reaches a stop codon on mRNA causing the binding of a release factor into the A site of the large subunit
- The release factor promotes breakdown of tRNA in the P site from mRNA. Bond between this tRNA and polypeptide is also broken down causing the release of the polypeptide chain
- Ribosomal subunits and other components dissociate - requires energy from two GTP molecules
Describe amino acid structure
Central carbon atom bonded to amino group, carboxylic group, H atom and R group
20 different R groups forming 20 different amino acids
R group determines amino acid structure and function
What is the 5’ end and 3’ end of amino acid chains?
N-terminus (amino end) - 5’ end
C-terminus (carboxyl end)- 3’ end
What is primary structure?
The sequence of amino acids in a polypeptide chain held together by peptide (covalent) bonds
What is secondary structure?
Held by weak hydrogen bonds to form alpha helix or Beta pleated sheets
What is tertiary structure?
Further folding and coiling to form complex 3D shape due to various side chain interactions
What is quaternary structure?
Association of multiple proteins forming a functional protein
Where are the ribosomes for translation found?
Free in the cytoplasm
How are endomembrane proteins translated?
The translation begins by ribosomes in the cytoplasm however the process is completed at fixed ribosomes on the RER for packaging.
How are ribosomes in the cytoplasm directed to the rough ER for the completion of translation of endomembrane proteins?
- Polypeptide synthesis begins
- Signal recognition particle binds to signal peptide (a part of chain already made)
- Signal recognition particle binds to receptor protein on ER membrane
- Signal recognition particle detaches and polypeptide synthesis resumes
- Signal-cleaving enzyme cuts off signal peptide
- Completed polypeptide folds into protein
What is signal peptide?
A length of polypeptide on the N terminus of protein which the signal recognition particle binds to allowing the complex to bind to the receptor protein of ER and hence move the polypeptide synthesis into the ER.
What is the purpose of post translational modifications?
Can confer activity (e.g. phosphorylation of enzyme)
Ability to interact with other molecules
Direct to particular locations
