ch.12

Central Dogma of Molecular Genetics (Francis Crick, 1958): Describes the flow of genetic information.
- DNA Replication: The process where DNA makes copies of itself; essential for cell division and hereditary transmission.
- Gene: A unit of heredity, represented by chromosomal DNA, that encodes the necessary information for creating proteins.
- Transcription: The synthesis of RNA from a DNA template to produce an RNA copy of a gene.
- Messenger RNA (mRNA): A type of RNA that serves as a temporary copy of a gene that can be translated into polypeptides.
- Translation: The process whereby the information in mRNA is used to synthesize polypeptides, which form functional proteins contributing to phenotypic traits in organisms.

Outline the relationship between DNA, RNA, and proteins:
- Transcription: DNA to RNA.
- Translation: RNA to Protein.
Major RNA Types:
- mRNA: Encodes amino acid sequences (generally monocistronic in eukaryotes).
- rRNA: Structural and functional components of ribosomes.
- tRNA: Transfers specific amino acids during translation.
- snRNA: Involved in splicing introns from pre-mRNA.
- miRNA: Regulates gene expression by interfering with mRNA stability.
- siRNA: Regulates gene expression and maintains genome integrity.
- Telomerase RNA: Part of the telomerase complex, necessary for telomere maintenance.

Gene Structure:
- Promoter: DNA sequence where RNA polymerase binds to initiate transcription.
- RNA-coding Sequence: The segments of DNA that are transcribed into RNA.
- Terminator: Sequence signaling the end of transcription.
Transcription Direction:
- 5' to 3' directionality in mRNA synthesis with specific untranslated regions (5' UTR and 3' UTR).
- The 5' UTR is critical for translation initiation, while the 3' UTR plays roles in mRNA stability and localization.

Initiation: RNA polymerase binds to the promoter, forming a closed complex before transitioning to an open complex.
Elongation: RNA polymerase synthesizes RNA by unwinding the DNA template and adding nucleotide triphosphates (NTPs) in the 5' to 3' direction.
Termination: RNA polymerase reaches the termination sequence, causing the release of the new RNA transcript.

Promoter Sequences in E. coli:
- -10 Sequence (Pribnow Box): 5′-TATAAT-3′
- -35 Sequence: 5′-TTGACA-3′
RNA Polymerase Enzymes:
- Core Enzyme: Main component responsible for RNA synthesis.
- Holoenzyme: Core enzyme plus sigma (σ) factor needed for initiation.

RNA Pol holoenzyme binds to the promoter via σ factor, forming a closed promoter complex.
Transition to an open promoter complex occurs when AT base-pairs in the -10 sequence separate.
σ factor is released, and elongation begins with the RNA Pol core enzyme.

Elongation Steps:
1. RNA Pol unwinds the DNA, utilizing helicase activity to synthesize RNA from the template strand.
2. The DNA rewinds behind the RNA polymerase, continuously elongating the RNA strand.

Termination Mechanisms:
1. Rho-dependent Mechanism:
2. ρ protein binds to the rho utilization (rut) sequence near the 3′ end of RNA.
3. A stem-loop forms due to inverted repeats, causing RNA Pol to pause.
4. ρ protein catches up, using helicase activity to separate the RNA-DNA hybrid, releasing the RNA transcript.
5. Rho-independent Mechanism:
6. RNA Pol transcribes a stem-loop region, which pauses transcription.
7. The A=U base-pairs break, leading to the release of the RNA transcript.

Promoter Elements:
- Core promoter commonly features a TATA box (-25 sequence).
- Enhancers may be found at variable locations relative to the promoter.

TFIID binds to TATA box and downstream promoter element (DPE).
RNA Pol II and general transcription factors (GTFs) bind to form a closed complex.
TFIIH unwinds DNA to create an open complex, simultaneously phosphorylating CTD of RNA Pol II.
Initiation concludes, and transcription begins as GTFs and mediator are released.

Allosteric Model: RNA Pol II dissociates post-transcription of the polyadenylation signal.
Torpedo Model: An exonuclease binds to the 5' end of the nascent RNA and digests it until it catches up with RNA Pol II, terminating transcription.

5' Capping: Addition of a 7-methylguanosine (m7G) cap to the 5' end of mRNA, facilitating splicing, nuclear export, and translation.
3' Polyadenylation: A tail of adenine nucleotides (up to 250 As) added to the 3' end enhancing RNA stability and translation efficiency.
Splicing: The process of removing introns and joining exons; can produce multiple mRNAs from a single gene via alternative splicing.
RNA Editing: Post-transcriptional modification of nucleotides in RNA molecules.

Splicing Process

Involves two transesterification reactions where introns are excised, forming a lariat structure while exons are ligated together.

Alternative Splicing

Allows differential protein production from a single mRNA based on which exons are included, leading to protein diversity across different cell types.

Promoter Elements
- Bacteria: Consist of -35/-10 sequences.
- Archaea: Features include TATA boxes and BRE.
- Eukaryotes: Complex promoter structures with TATA boxes and additional elements.
RNA Polymerases: Bacteria and Archaea use a single type, while Eukaryotes utilize three.
Splicing and Capping: Eukaryotes primarily perform these modifications, while they are rare in prokaryotes.

A method used to detect specific RNA sequences in a sample through gel electrophoresis followed by hybridization with labeled probes.

Used to analyze protein-DNA interactions; slower migration in gel indicates bound proteins to DNA.

Involves using DNase I to digest unprotected DNA, allowing identification of the protein-binding regions in the DNA by observing the cleavage pattern of end-labeled DNA fragments.