The Nature of GenesEarly ideas to explain how genes work came from studying human diseases. | Archibald Garrod proposed that patients with the disease alkaptonuria lacked a particular enzyme. | Beadle and Tatum studied Neurospora crassa.
They looked for fungal cells lacking specific enzymes. | Beadle and Tatum results was that each mutated enzyme disrupted one key enzyme in the metabolic pathway. |
Prokaryotic TranscriptionProkaryotic Transcription: Single RNA polymerase
- Initiation of mRNA synthesis does not require a primer | Prokaryotic Transcription requires a Promoter, Start Site, and a termination site. | Transcription occurs in three major stages:
- Initiation
- Elongation
- Termination | Initiation: RNA polymerase binds to the promoter | Promoter: Forms a recognition and binding site for the RNA polymerase.
- Found upstream of the start site.
- Not transcribed.
- Asymetrical: indicate site of initiation and direction of termination. | Elongation: RNA transcript grows in the 5'-to-3' direction as ribonucleotides are added.
- Transcription bubble: contains RNA polymerase, DNA template, and growing RNA transcript.
- After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble. | Termination: Marked by sequence that signals "stop" to polymerase.
- Causes the formation of phosphodiester bonds to cease.
- RNA-DNA hybrid within the transcription bubble dissociates.
- RNA polymerase releases the DNA.
- DNA rewinds. | Hairpin in RNA causes RNA polymerase to pause | U:A base pairs weaken the DNA/RNA bonding. | Prokaryotic transcription is coupled to translation
- mRNA begins to be translated before transcription is finished. |
Frameshift mutations
- Addition or deletion of a single base
- Much more profound consequences
- Alter reading frame downstream
- Triplet repeat expansion mutation
| Hunting disease
Repeat unit is expanded in the disease allele relative to the normal |
| | Transcription and TranslationThe Central Dogma | Information only flows from: DNA-->RNA-->protein
First described by Francis Crick. | Transcription | DNA--> RNA
- DNA-directed synthesis of RNA
- Only template strand of DNA used
- T in DNA replaced by U in RNA.
- mRNA used to direct synthesis of polypeptides. | Translation | - Synthesis of polypeptides.
- Takes place at ribosome.
- Requires several kinds of RNA. | RNA | All synthesized from DNA template by transcription
- Messenger RNA (mRNA).
- Ribosomal RNA (rRNA).
- Transfer RNA (tRNA).
- Small nuclear RNA (snRNA)
- Signal recognition particle RNA (SRP RNA).
- Micro-RNA (miRNA). |
Eukaryotic Transcription3 different RNA polymerase!! | RNA polymerase I: Transcribes rRNA. | RNA polymerase II: treanscribes mRNA and some snRNA. | RNA polymerase III: transcribes tRNA and some other small RNAs | Each RNA polymerase recognizes it own promoter. | Initiation of transcription: Requires a series of transcrption factors (helper).
- Transcription factors: Necessary to get the RNA polymerase II enzyme to a promoter* and to initiate gene expression. | Elongation: RNA transcribed from the DNA template. | Termination not as well defined. |
Initiation of trancription
- Transcription factors bind to a promoter region and recruit RNA polymerase.
- Forms the initation complex. Protein TargetingIn eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER). | Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition (SRP). |
- The signal sequence and SRP are recognized by RER receptor proteins
- Docking holds ribosome to RER
- Beginning of the protein-trafficking pathway
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Mutation: Altered GenesPoint mutations | alter a single base | Base substitution | substitute one base for another | Siletn mutation | same amino acid inserted | Missense mutation | changes amino acid inserted
- Transitions
- Transversions | Nonsense mutations | changed to stop codon |
| | The Genetic CodeFrancis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order.
- They introduced single nucleotide insertions or deletions and looked for mutations (Frameshift mutations) | A Codon is a block of three DNA nucleotides corresponding to an amino acid, | Spaced Codons: Codon sequence in a gene punctuated. | Unspaced Codons: codons adjacent to each other.
- Marshall Nirenberg identified the codons that specify each amino acid. | Stop Codon: 3 codons (UAA, UGA, UAG) used to terminate translation | Start Codon: Codon (AUG) used to signify the start of translation | Code is degenerate: Some amino acids are specified by more that one codon. | Code practically unicersal: Strongest evidence that all living things share common ancestry.
- Advanced in genetic engineering.
- Mitochondria and cloroplast have some differences in "stop" signals. |
mRNA modificationsIn eukaryotes the primary transcript must be modified to become mature mRNA | Addition of a 5' cap Protects nucleotides from getting lost, from degradation.
- Involved in translation initiation. | Addition of a 3' poly-A tail Created by poly-A polymerase, protection from degradation
- Puts whole string of A's (AAA) to protect! | Removal of noncoding sequences (introns): Pre-mRNA splicing done by spliceosome.
- Cut it out to get rid of it!!! |
tRNA charging reactionEach aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs. | Charged tRNA has an amino acid added using the energy from ATP.
-Can undergo peptide bond formation without additional energy. | Ribosomes do not verify amino acid attached to tRNA. | The ribosome has multiple tRNA binding sites: | P site: binds the tRNA attached to the growing peptide chain | A site: binds the tRNA carrying the next amino acid. | E site: binds the tRNA that carried the last amino acid, tRNA exits ribosome. | The ribosome has two primary functions
- Decode the mRNA.
- Form peptide bonds. | Peptidyl transferase:
- Enzymatic component of the ribosome.
- Forms peptide bonds between amino acids. |
Chromosomal mutationsChang the structure of a chromosome | Deletions: part of chromosome is lost | Duplication: part of chromosome is copied | Inversion: part of chromosome in reverse order | Translocation: part of chromosome is moved to a new location |
| | Eukaryotic pre-mRNA splicingIntrons | non-coding sequences | Exons | sequences that will be translated | Small ribonucleoprotein particles (snRNPs "snurps") | Looks for introns and exons and recognizes it. | Spliceosomes | responsible for removing introns |
snRNPs cluster with other proteins to form spliceosome tRNA and RibosomestRNA moleules carry amino acids to the ribosome for incorporation into a polypeptide.
- Aminoacyl-tRNA synthetase add amino acids to the acceptor stem of tRNA.
- Anticodon loop contains 3 nucleotides complementary to mRNA codons. |
TranslationProcess by which the mRNA transcript is read by the ribosomes and used to make a polypeptide.
Occurs in 3 main stages:
- Initiation
- Elongation
- Termination | There are some important differences between translation in prokaryotes and eukaryotes. | In prokaryotes, initiation complex includes Initiator tRNA charged with N-formylmethionine {[nl}}- Small ribosomal subunit
- mRNA strand |
- Ribosome binding sequence (RBS) of mRNA positions small subunit correctly.
- Large subunit now added.
- Initiator tRNA bound to P site with A site empty.
| Initiations in eukaryotes similar except:
- Initiating amino acid is methionine.
- Lack of an RBS – small subunit binds to 5′ cap of mRNA. | Elongation adds amino acids
- 2nd charged tRNA can bind to empty A site
- Requires elongation factor called EF-Tu to bind to tRNA and GTP
- Peptide bond can then form.
- Addition of successive amino acids occurs as a cycle. |
- There are fewer tRNAs than codons
- Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon
- This allows fewer tRNAs to accommodate all codons
| Termination
- Elongation continues until the ribosome encounters a stop codon
- Stop codons are recognized by release factors which release the polypeptide from the ribosome |
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