Week 9

Molecular Structure and Chemical Properties of RNA

• Intermediary Role of RNA: While DNA stores information in the nucleus (in eukaryotes), proteins are synthesized on ribosomes in the cytoplasm. RNA serves as the intermediary molecule transferring genetic information between these locations.

• Chemical Comparison: RNA vs. DNA:

  • Sugar Component: RNA contains the five-carbon sugar ribose. Ribose has a reactive hydroxyl group ($OH$) on the $2'$ carbon, whereas deoxyribose in DNA has a hydrogen atom at the same position. This additional hydroxyl group contributes to RNA being less stable than DNA.

  • Bases: RNA utilizes Uracil ($U$) instead of Thymine ($T$). Uracil and Thymine differ in that Thymine has a methyl group ($CH_3$) at its number 5 carbon, while Uracil has a hydrogen atom ($H$). The hydrogen-bonding groups are identical, allowing Uracil to pair with Adenine ($U\text{--}A$) similarly to $T\text{--}A$ pairs.

  • Strand Polarity and Ends: RNA chains are linked by phosphodiester bonds. A significant difference is that the $5'$ end of a DNA strand is typically a monophosphate, whereas the $5'$ end of an RNA molecule is typically a triphosphate.

• Physical Distinctions:

  • Length: RNA molecules are significantly shorter than DNA. A typical RNA used in protein synthesis contains a few thousand nucleotides; a typical DNA molecule consists of millions or tens of millions.

  • Conformation: DNA is primarily double-stranded, while most cellular RNA is single-stranded. However, single-stranded RNA can fold upon itself (forming hairpins) to create complex three-dimensional structures with double-stranded regions. These structures can rival proteins in complexity and enable RNA to act as a catalyst (ribozyme).

  • formation storage, while proteins are more versatile for catalysis and cellular functions.

Gene Regulation (watch video again did not understand)

• a set of genes that are turned on in that particular cell type to give it its traits

• gene regulation can occur at any point in the journey from the DNA to the final protein product

• there is positive and negative transcription regulation

positive regulation

• positive regulation means an additional factor is required to induce transcription

• activator protein (transcription factor) that is required for RNA polymerase complex to bind to this particular gene and induce transcription

• if activator is not present or cannot bind the RNA polymerase is recruited and therefore no transcription can occur

negative regulation

• RNA polymerase complex can bind on its own so default is that transcription is occuring

• if a different type of transcription factor (repressor) is present in the cell, and is able to bind to the regulatory element that inhibits the recruitment of RNA polymerase and therefore transcription does not occur

Regulated expression

constitutive expression

lac operon

The Mechanism and Stages of Transcription

Key words

• enhancer sequences

• promoter sequences

• transcriptional activator proteins

• general transcription factors

• 
  

• Basic Process: Transcription uses DNA as a template to synthesize a complementary RNA transcript. The template DNA strand is read in the $3' \rightarrow 5'$ direction, while the RNA transcript is synthesized in the $5' \rightarrow 3'$ direction. The nontemplate strand is not transcribed. transcription is essential to producing proteins

• mRNA translated by the ribosomes to form proteins

• RNAs polymerase binds at the promoter region that forms a compelx with the DS DNA and that enzymatic activity enable the DS DNA to split into two SS DNA strands, allowing the RNA polymerase to access the template strand. This process initiates transcription, leading to the elongation of the RNA molecule as it synthesizes complementary nucleotides. . one split the polymerase initiated the production of RNA transcript

• RNA transcript is happening from 5’ to 3’ end

• before RNA is exported to the cytoplasm, 3 things occurs ONLY IN EUKAYOTES this makes up the mature mRNA as apposed to the pre mRNA

• 1. 5’ methylated cap is attached tot eh 5’ end of the mRNA molecule

• 2. at the 3’ end there is the enzymatic addition of a string of evenings called the PolyA tail

• 3. regions called introns are removed from the RNA molecule to form the mature RNA ( SPLICING)

SPLICING

• starts w primary transcript

• proteins form a ‘spliceosome’ bind to RNA molecule at various locations

• some at the boundary between exon and intron and other nudn int he middle of the intron and reaction occurs where site at the 5’ slice site is attacked releasing the intronic sequence that forms a lariat (loop) this stretch is the then degraded into individual nucleotides

• spliceosome is release and remains are the exons adjacent to the rna MOLECULE

• The do this as alternative splicing allows for more than one protein product to be produced from a single gene

• some introns may not be sliced out or exons may not be included and this allows more than one type of protein to be produced from one primary transcript enabling genetic diversity

• Stages of Transcription:

  1. Initiation: RNA polymerase and associated proteins (transcription factors) bind to double-stranded DNA, separate the strands, and begin transcription at the promoter sequence. DNA enhancers are recognised by proteins in the nucleous (transcriptional activator proteins) and will bind to that DNA

  2. Elongation: Ribonucleoside triphosphates (rNTPs) are added successively to the $3'$ end of the growing RNA transcript.

  3. Termination: RNA polymerase encounters a terminator sequence in the template strand, stops transcription, and releases the transcript.

• Polymerization Chemistry:

  • The RNA polymerase orients the $3'$ hydroxyl ($OH$) of the growing strand to attack the innermost phosphate bond of the incoming triphosphate.

  • Cleavage of this high-energy phosphate bond drives the reaction. The release and subsequent cleavage of a pyrophosphate molecule ($PP_i$) by another enzyme make the polymerization reaction irreversible.

• RNA Polymerase Characteristics:

  • It is a large multiprotein complex that functions as a molecular machine to open, transcribe, and close DNA.

  • Accuracy: Incorporates fewer than $1$ incorrect nucleotide per $10,000$.

  • Proofreading: Unlike DNA polymerase, RNA polymerase lacks an error-correcting function. Evolutionary logic dictates that transcriptional errors are less critical as they only affect transient transcripts, whereas DNA errors are passed to future generations.

Transcriptional Regulation in Prokaryotes: The Lac Operon

• because in prokaryotes there is no separation from nucleus from cytoplasm

• translation can begin before transcription is finished

• while transcription is still happening a ribosome can already bind to the new RNA molecule and start producing a polypeptide from it

• cannot happen in eukaryotes as transcription and translation happen in different places, transcription occurs in nucleus and translation in the cytoplasm

An operon:

• two or ore genes that are transcribed together and produce products that are involved in a combined biological process

• Promoter Recognition: In bacteria, promoter recognition is mediated by the sigma factor, a protein that facilitates RNA polymerase binding to specific sequences (often at $-10$ and $-35$ base pairs relative to the start site). Sigma factor dissociates after initiation.

• The Operon Model: Prokaryotes often organize functionally related genes together. An operon consists of a promoter, an operator, and multiple structural genes transcribed into a single polycistronic mRNA.

• Regulation Mechanisms:

  • Negative Regulation: A repressor protein binds to the operator to prevent transcription. In the lactose ($lac$) operon, the product of the lacI gene acts as the repressor.

  • Positive Regulation: A transcriptional activator protein must bind to DNA to recruit RNA polymerase. Example: CRP-cAMP complex.

• Case Study: The Lactose ($lac$) Operon:

  • Structural Genes: $lacZ$ (encoding $\beta$-galactosidase to cleave lactose) and $lacY$ (encoding lactose permease to transport lactose).

  • Induction: In the absence of lactose, the repressor binds the operator ($lacO$). When lactose is present, allolactose (an isomer of lactose) acts as an inducer, binding to the repressor and changing its shape so it cannot bind the operator.

  • Glucose Preference: The cell uses glucose before lactose. When glucose is low, levels of the molecule cAMP are high. cAMP binds to CRP (cAMP receptor protein), which then binds near the promoter to stimulate transcription. If glucose is high, cAMP is low, and the $lac$ operon is not transcribed at high levels even if lactose is present.

Transcriptional Regulation in Eukaryotes

• General Transcription Factors (GTFs): At least six proteins must assemble at the promoter (e.g., the TATA box, situated $25\text{--}30$ nucleotides upstream of the start site) to attract RNA polymerase.

• Transcription Activator Proteins: These bind to specific DNA sequences called enhancers. They recruit a mediator complex, which in turn recruits the RNA polymerase complex (Pol II for protein-coding genes). Because enhancers can be distant, DNA may loop to facilitate contact.

• Silencers: DNA sequences where regulatory transcription factors bind to repress transcription.

• Combinatorial Control: Transcription depends on the specific combination of regulatory factors present in a cell.

RNA Processing in Eukaryotes

• Coupling: In prokaryotes, transcription and translation are coupled (co-occurring). In eukaryotes, the nuclear envelope separates them, necessitating RNA processing within the nucleus.

• 5' Cap: A 7-methylguanosine is attached to the $5'$ end via an unusual triphosphate bridge between the $5'$ carbons of both ribose sugars. It is essential for ribosome recognition and translation initiation.

• Polyadenylation: A string of approximately $250$ Adenine nucleotides (the poly(A) tail) is added to the $3'$ end. This tail assists in nuclear export and stabilizes the transcript against degradation.

• RNA Splicing: The removal of noncoding introns and joining of coding exons. This is catalyzed by the spliceosome (a complex of RNA and protein, including snRNA).

• Alternative Splicing: Primary transcripts can be spliced in different ways to produce multiple protein products from one gene. Example: The insulin receptor transcript. In liver cells, Exon 11 is included (low-affinity receptor); in skeletal muscle, Exon 11 is removed (high-affinity receptor), allowing better glucose uptake.

• RNA Editing: Enzymes chemically modify bases (e.g., Adenosine to Inosine; Cytosine to Uracil). Example: Apolipoprotein B. Liver transcribes a full-length protein; intestinal cells edit a Cytosine to a Uracil ($UAA$ stop codon), resulting in a protein half as long for lipid absorption.

Noncoding RNAs and Additional Regulatory Layers

• Abundant RNA Types: In mammalian cells, approximately $80\%$ of RNA is Ribosomal RNA (rRNA) and $10\%$ is Transfer RNA (tRNA).

• Small Regulatory RNAs: miRNA (microRNA) and siRNA (small interfering RNA) form hairpin structures, are cleaved into $20\text{--}25$ bp fragments, and incorporated into the RISC (RNA-induced silencing complex) to degrade mRNA or inhibit translation.

• Translational Regulation: Factors include the folded structure of the $5'$ UTR, the distance to the $AUG$ initiation codon, and binding of proteins to the $3'$ UTR or poly(A) tail.

• Post-translational Modification:

  • Chaperones: Assist in proper protein folding.

  • Chemical Additions: Glycosylation (sugars) and Phosphorylation (reversibly adding phosphate groups to serine, threonine, or tyrosine to toggle activity).

  • Degradation: Marking proteins for destruction (e.g., cyclins).

Epigenetics and Chromatin Remodeling

• Chromatin Remodeling: Repositioning nucleosomes to expose DNA for transcription.

• Histone Modifications: The "histone code" involves adding methyl ($Me$) or acetyl ($Ac$) groups to lysine residues on histone tails.

• DNA Methylation: Heavy methylation of CpG islands (cytosine-guanine clusters near promoters) usually represses transcription.

• Imprinting: Sex-specific silencing of genes. Example: Size differences in Ligers (male lion + female tiger) vs. Tigons (male tiger + female lion) result from the inheritance of growth-related imprinted genes.

• X-Inactivation: To achieve dosage compensation, mammals inactivate one X chromosome in females. The Xist gene produces a noncoding RNA that coats the chromosome, recruiting silencing factors. This creates a phenotypic mosaic (e.g., calico cats).

Environmental and Lifestyle Impacts

• Feedback Mechanisms: Environment and lifestyle (diet, exercise, stress) influence regulatory transcription factors.

• Dietary Example: High fat/cholesterol intake affects the activity of enzymes and the transcription of genes encoding those enzymes.

• Disease Management: While Mendelian traits (Cystic Fibrosis) are caused by single mutations, lifestyle choices (exercise, avoiding smoking) still significantly influence the health outcomes of affected individuals.