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Early ideas to explain how genes work came from studying human diseases.
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Archibald Garrod proposed that patients with the disease alkaptonuria lacked a particular enzyme.
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Beadle and Tatum studied Neurospora crassa.
They looked for fungal cells lacking specific enzymes. |
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Beadle and Tatum results was that each mutated enzyme disrupted one key enzyme in the metabolic pathway.
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Prokaryotic Transcription: Single RNA polymerase
- Initiation of mRNA synthesis does not require a primer |
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Prokaryotic Transcription requires a Promoter, Start Site, and a termination site.
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Transcription occurs in three major stages:
- Initiation - Elongation - Termination |
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Initiation: RNA polymerase binds to the promoter
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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. |
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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. |
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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. |
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Hairpin in RNA causes RNA polymerase to pause
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U:A base pairs weaken the DNA/RNA bonding.
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Prokaryotic transcription is coupled to translation
- mRNA begins to be translated before transcription is finished. |
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- Addition or deletion of a single base - Much more profound consequences - Alter reading frame downstream - Triplet repeat expansion mutation |
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Hunting disease
Repeat unit is expanded in the disease allele relative to the normal |
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The Central Dogma
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Information only flows from: DNA-->RNA-->protein
First described by Francis Crick. |
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Transcription
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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. |
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Translation
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- Synthesis of polypeptides.
- Takes place at ribosome. - Requires several kinds of RNA. |
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RNA
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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). |
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3 different RNA polymerase!!
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RNA polymerase I: Transcribes rRNA.
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RNA polymerase II: treanscribes mRNA and some snRNA.
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RNA polymerase III: transcribes tRNA and some other small RNAs
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Each RNA polymerase recognizes it own promoter.
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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. |
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Elongation: RNA transcribed from the DNA template.
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Termination not as well defined.
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In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER).
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Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition (SRP).
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- 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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Point mutations
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alter a single base
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Base substitution
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substitute one base for another
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Siletn mutation
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same amino acid inserted
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Missense mutation
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changes amino acid inserted
- Transitions - Transversions |
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Nonsense mutations
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changed to stop codon
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Francis 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) |
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A Codon is a block of three DNA nucleotides corresponding to an amino acid,
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Spaced Codons: Codon sequence in a gene punctuated.
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Unspaced Codons: codons adjacent to each other.
- Marshall Nirenberg identified the codons that specify each amino acid. |
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Stop Codon: 3 codons (UAA, UGA, UAG) used to terminate translation
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Start Codon: Codon (AUG) used to signify the start of translation
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Code is degenerate: Some amino acids are specified by more that one codon.
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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. |
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In eukaryotes the primary transcript must be modified to become mature mRNA
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Addition of a 5' cap Protects nucleotides from getting lost, from degradation.
- Involved in translation initiation. |
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Addition of a 3' poly-A tail Created by poly-A polymerase, protection from degradation
- Puts whole string of A's (AAA) to protect! |
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Removal of noncoding sequences (introns): Pre-mRNA splicing done by spliceosome.
- Cut it out to get rid of it!!! |
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Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs.
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Charged tRNA has an amino acid added using the energy from ATP.
-Can undergo peptide bond formation without additional energy. |
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Ribosomes do not verify amino acid attached to tRNA.
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The ribosome has multiple tRNA binding sites:
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P site: binds the tRNA attached to the growing peptide chain
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A site: binds the tRNA carrying the next amino acid.
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E site: binds the tRNA that carried the last amino acid, tRNA exits ribosome.
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The ribosome has two primary functions
- Decode the mRNA. - Form peptide bonds. |
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Peptidyl transferase:
- Enzymatic component of the ribosome. - Forms peptide bonds between amino acids. |
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Chang the structure of a chromosome
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Deletions: part of chromosome is lost
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Duplication: part of chromosome is copied
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Inversion: part of chromosome in reverse order
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Translocation: part of chromosome is moved to a new location
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Introns
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non-coding sequences
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Exons
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sequences that will be translated
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Small ribonucleoprotein particles (snRNPs "snurps")
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Looks for introns and exons and recognizes it.
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Spliceosomes
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responsible for removing introns
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tRNA 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. |
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Process by which the mRNA transcript is read by the ribosomes and used to make a polypeptide.
Occurs in 3 main stages: - Initiation - Elongation - Termination |
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There are some important differences between translation in prokaryotes and eukaryotes.
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In prokaryotes, initiation complex includes Initiator tRNA charged with N-formylmethionine {[nl}}- Small ribosomal subunit
- mRNA strand |
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- Ribosome binding sequence (RBS) of mRNA positions small subunit correctly. - Large subunit now added. - Initiator tRNA bound to P site with A site empty. |
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Initiations in eukaryotes similar except:
- Initiating amino acid is methionine. - Lack of an RBS – small subunit binds to 5′ cap of mRNA. |
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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. |
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- 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 |
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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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