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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