Protein Synthesis: Transcription & Translation

Protein Synthesis: Transcription

• Transcription definition: RNA synthesis from a DNA template, converting DNA-encoded genetic information into a complementary RNA sequence.
• DNA (large) relies on mRNA to carry its protein-making instructions outside the nucleus.
• Transcription and translation are the core stages of protein creation.

Transcription and Translation Defined

• Transcription: DNA is read to produce a strand of mRNA.
• Translation: mRNA is read to produce an amino acid chain in the ribosome
• Transfer RNA (tRNA) carries specific amino acids.
• mRNA degrades to prevent the overproduction of proteins.
• RNA polymerase unwinds DNA to expose about 12 base pairs at a time.
• RNA polymerases separate DNA strands to prepare the template for RNA creation.
• DNA splits into a template strand and a copying strand.
• RNA polymerase adds complementary ribonucleotides to the DNA template strand, forming an RNA molecule.
• Non-coding mRNA strands (introns) are removed, while coding strands (exons) are spliced together.
• RNA polymerase detaches, the DNA double helix reforms, and mRNA exits via nuclear pores into the cytoplasm.
• RNA polymerases correct errors during synthesis.

Hydrogen Bonding and Base Pairing During

• Complementary Base Pairing rules: Adenine (A) pairs with Uracil (U) in RNA; Cytosine (C) pairs with Guanine (G).
• Sense strand: DNA strand with the same sequence as the RNA transcript, except with T instead of U.
• Antisense strand: The template strand, which is complementary to the RNA transcript, used for RNA synthesis.

DNA Stability During Transcription

• DNA's double helix structure provides stability and protection.
• Hydrogen bonds between bases hold the strands together.
• Supercoiling enhances stability.
• Repair mechanisms maintain genetic integrity.

Transcription and Gene Expression Defined

• Gene expression definition: information from a gene is used to synthesize a functional gene product (typically a protein).
• The major steps are transcription, RNA processing, and translation.
• Specific gene expression patterns lead to cell differentiation.
• Transcription controls gene expression by determining which genes are expressed and when.

Protein Synthesis: Translation

• Translation definition: Polypeptides (proteins) are synthesized from mRNA sequences.
• Translation occurs on ribosomes in the cytoplasm.

Roles of mRNA, Ribosomes, and tRNA

• mRNA's role is to carry genetic code from DNA to ribosomes, acting as the template for translation.
• Ribosomes are the location of protein synthesis, composed of rRNA and proteins, facilitating the linkage of amino acids.
• tRNA transfers amino acids to the ribosome and has an anticodon that pairs with mRNA codons.
• mRNA is linear, with a 5' cap and a 3' poly-A tail.
• tRNA has a cloverleaf shape, including an amino acid attachment site and an anticodon, with three exposed bases matching mRNA codons.
• The ribosome structure is made of small and large subunits, with tRNA binding sites A (aminoacyl), P (peptidyl), and E (exit).

Base Pairing Between tRNA and mRNA

• A codon is a three-nucleotide sequence on mRNA coding for an amino acid.
• An anticodon is the complementary sequence on tRNA.
• Hydrogen bonds form between the codon on mRNA and anticodon on tRNA.

Features of the Genetic Code

• A triplet code is where a codon codes for each of the 20 amino acids.
• The genetic code also includes a degenerate and universal codon.
• Degenerate codon: Multiple codons can code for the same amino acid.
• Universal codon: The genetic code is largely the same across different organisms.

Genetic Code Table

• Genetic code tables are utilized to translate mRNA or DNA sequences into amino acid sequences.

Translation Elongation

• Codon recognition: tRNA anticodon pairs with the mRNA codon at the A site.
• Bond formation: A peptide bond forms between amino acids in the P and A sites.
• Translocation: The ribosome moves along the mRNA, shifting the tRNA from A to P and releasing the empty tRNA from the E site.

Mutations that Change Protein Structure

• A gene mutation definition: A change in the nucleotide sequence of a gene.
• Cause: A single nucleotide mutation in the β-globin gene leads to the production of hemoglobin S (HbS) instead of hemoglobin A (HbA).
• RNA Sequence Differences: The mutation results in a different mRNA sequence for HbS compared to HbA.
• HbS contains valine instead of glutamic acid at the sixth position.
• Insoluble haemoglobin is unable to carry oxygen effectively.
• Fibrous haemoglobin strands change red blood cell shape to sickle shaped.
• Consequences: Sickle cells may clot capillaries, blocking blood, and also are destroyed rapidly, leading to anemia.
• Symptoms include pain, increased infection risk, and organ damage from poor blood flow.

Directionality of Transcription and Translation

• The 5' end of RNA has a phosphate group; the 3' end has a hydroxyl group.
• Covalent bonds form between nucleotides, linking the 5' phosphate to the 3' hydroxyl of the growing RNA strand.
• RNA polymerases synthesize RNA in the 5' to 3' direction.
• Ribosomes move along mRNA in the 5' to 3' direction during translation.

Transcription Initiation at the Promoter Defined

• Promoters are specific DNA sequences upstream of a gene that signal the start of transcription.
• Process: RNA polymerase binds to the promoter to start transcription. Transcription factors assist RNA polymerase.
• Activator sequences enhance transcription while repressor sequences inhibit transcription.

Non-Coding DNA

• Coding sequences code for proteins; non-coding sequences do not.
• Regulatory elements control gene expression.
• Introns are removed during RNA processing.
• Telomeres protect chromosome ends.
• Centromeres are essential for chromosome segregation.
• Transposable elements can move and influence gene expression.

Post-Transcriptional Modification in Eukaryotic Cells

• Location and timing: Modifications occur in the nucleus after transcription.
• 5' Cap: A modified guanine nucleotide added to the 5' end, aiding in stability and ribosome recognition.
• Poly-A Tail: Adenine nucleotides added to the 3' end, enhancing mRNA stability and export from the nucleus.
• Introns: Non-coding regions removed during RNA splicing.
• Exons: Coding regions are joined to form the mature mRNA.
• RNA Splicing: Involves intron removal and exon joining.

Alternative Splicing

• Alternative splicing is where different exon combinations create multiple mRNA variants from a single gene.
• This process increases protein diversity.

Translation

• mRNA Binding: The small ribosomal subunit binds to the mRNA.
• tRNA Binding: Initiator tRNA binds to the start codon (AUG) on the mRNA.
• Large Subunit Joining: The large ribosomal subunit assembles, forming the complete ribosome.

Modification of Polypeptides Defined

• Phosphorylation: Addition of phosphate groups.
• Glycosylation: Addition of carbohydrate groups.
• Proteolytic Cleavage: Cutting polypeptide chains into active forms.
• Preproinsulin is cleaved to form proinsulin, is folded and cleaved again to functional insulin.

Recycling of Amino Acids by Proteasomes

• Proteins may breakdown due to regulatory needs or damage.
• Proteasomes degrade proteins, recycling amino acids for new synthesis.

Gene Mutations

• A mutation definition is: A permanent change in the nucleotide sequence of a gene or a chromosome in a cell or a virus, which can lead to changes in the structure and function of proteins.
• Silent mutation: Results in a base change but still codes for the same amino acid.
• Base Substitution: A single nucleotide is replaced by another, which can lead to various outcomes.
• Insertion: One or more bases are added into the DNA sequence.
• Deletion: One or more bases are removed from the DNA sequence.
• Insertions and deletions are more dangerous because all the codons are shifted, causing gene shift (frameshift mutation).

Base Substitutions and SNPs (Single Nucleotide Polymorphisms)

• SNPs are variations in a single base at a specific genomic position that are common in the population, potentially resulting in a different amino acid.
• SNPs are specific, while base substitution is any base swap.
• Coding sequence mutations can alter the resulting protein's function.
• Non-coding mutations may not affect protein function but can affect gene regulation.
• Genetic code degeneracy reduces mutation impact as some substitutions don't alter the protein.
• Types: Same-sense (silent), nonsense, and missense mutations.
• Silent: No changes in the amino acid sequence due to code redundancy.
• Nonsense: Creates a premature stop codon, truncating proteins.
• Missense: Results in a different amino acid, altering function.

Insertions and Deletions

• A frameshift mutation happens here.
• Frameshift mutations alter the codon reading frame.
• Polypeptide Structure: Insertions and deletions can significantly alter amino acid sequences and create nonfunctional proteins.

Causes of Gene Mutation

• Spontaneous Mutations: Occur naturally during DNA replication or repair.
• Induced Mutations: Caused by external factors like radiation or chemicals.
• A mutagen definition: An agent that causes changes in DNA, increasing the frequency of mutations.
• Forms of mutagens: High energy electromagnetic radiation like ultraviolet light, x-rays and gamma-rays.

Randomness in Gene Mutation

• Impact of Randomness: Mutations occur randomly, contributing to genetic diversity
• Cytosine has highest probability of mutating out of all the bases.
• Lack of Deliberate Change: No natural mechanisms are known to deliberately alter DNA sequences; mutations arise as random events.

Mutation Impact on Germ Cells and Somatic Cells

• Germ Cells: Develop into gametes that pass mutations to offspring, so alleles will be present in the zygote after fertilization.
• Somatic Cells: Non-reproductive cells that do not pass mutations on.
• Germ Cell Mutations: heritable, contributing to evolutionary changes and genetic disorders.
• Somatic Cell Mutations: Affect the individual organism but are not inherited.

Mutation and Genetic Variation

• Genetic variation is the differences in DNA sequences among individuals.
• Gene mutations are the origin of new alleles.
• Types of Gene Mutations: Beneficial, Neutral, Harmful Mutations.
• Beneficial Mutations: Enhance survival or reproductive success- natural selection.
• Neutral Mutations: Have no significant effect on fitness.
• Harmful Mutations: Detrimental to survival or reproduction.
• Most mutations are neutral as they occur in non-coding sections of DNA.

Gene Knockout

• Gene knockout definition: When a targeted gene is inactivated and disabled, this allows the investigation of its function.
• Gene knockout only works when it is an embryo.
• They are performed on mice, fruit flies, and zebrafish.
• Gene Knockout Methods is to target a specific gene to interrupt its function. Cells with the knockout are selected for analysis.

CRISPR-Cas9

• Design of gRNA: A guide RNA (gRNA) is designed to match the target a specific DNA sequence that is to be edited. This gRNA is crucial for directing the Cas9 enzyme to the correct location in the genome.
• The gRNA binds to the Cas9 enzyme, forming a ribonucleoprotein complex.
• Target DNA Recognition: The CRISPR-Cas9 complex is introduced into the cell, where the gRNA hybridizes with its complementary DNA sequence in the genome.
• The CRISPR-Cas9 can knock out a gene or knock a gene into a gene that was cut in the DNA.
• DNA Cleavage: Cas9 induces a double-strand break in the DNA at the target site.
• DNA Repair: The cell's natural repair mechanisms are activated.
• Non-Homologous End Joining (NHEJ): Often results in insertions or deletions (indels) that can disrupt the gene.
• Homology-Directed Repair (HDR): If a donor template is provided, it can be used to incorporate new genetic material.
• Uses: Medical research, gene therapy, agricultural and industrial biotechnology.
• CRISPR-Cas9: remove genes from mosquitos that have malaria. It can also increase drought or pest resistance in crops.
• Ethical Implications: Safety concerns, germline editing, equity, access, and biodiversity.

Conserved Sequences in Genes

• Conserved sequences: Regions that remain unchanged across species, showing functional importance.
• Highly conserved sequences are genes identical or similar over long evolution periods.
• Common Examples: Enzymes and Ribosomal RNA
• Functional Constraint Hypothesis: Conserved sequences are required for biological functions, which are maintained through natural selection.

Gene Expression and Phenotype

• Genotype: Genetic makeup of an organism.
• Phenotype: Observable characteristics resulting from the interaction of its genotype with the environment.
• Gene expression is the use of gene information to synthesize a functional gene product, typically a protein.
• Gene Expression: transcription, RNA processing, translation, post-translational modifications.

Regulation of Transcription

• Transcription Factors: Proteins that bind to promoter DNA sequences and affect transcription.
• Transcription initiator complex → several protein’s + RNA polymerase → distant activator complex bound to DNA initiates transcription
• Promoters are binding sites for RNA polymerase near the start of a gene.
• Enhancers: DNA sequences far from the gene that increase transcription.
• Activators: Proteins binding to enhancer DNA sequences and increase the transcription by helping the RNA polymerase to bind to the promoter.
• Suppressors (or Silencers): DNA sequences that decrease the likelihood of transcription when bound by repressor proteins.
• Repressors: Proteins that bind to promoter or enhancer sequences and block RNA polymerase binding.

mRNA Degradation and Translation Regulation

• The length of the poly-A tail on mRNA will affect its stability.
• Longer tails will enhance mRNA stability, while shorter tails lead to increased degradation by nucleases.
• mRNA regulates protein synthesis, allowing cells to respond dynamically.

Epigenesis and the Epigenome Defined

• Epigenesis: Distinct cell development/functions through changes in gene expression.
• Results from an interaction between genes and their environments.
• Epigenetic tags are chemical markers attached to DNA or histone proteins that influence the transcription of genes
• Epigenetics is the study of changes in organisms caused by modifications in the genes.
• Epigenome: The complete set of epigenetic modifications on the genetic material of a cell.

Genome, Transcriptome, and Proteome

• Genome: The organism’s complete DNA set.
• Transcriptome: Total RNA transcripts produced from the genome, reflecting gene expression.
• Proteome: Entire set of proteins expressed, indicating functional activity.

Methylation and Acetylation in Gene Expression

• Methyl groups are epigenetic tags.
• Methylation: Methyl groups are epigenetic tags that wind histone proteins tightly/inhibit enzymes to move along DNA, or attached to cysteine nucleotides in a promoter region repress promoters to prevent the genes from being transcribed, repressing gene expression.
• Acetylation: Addition of acetyl groups to histones leads to activation.
• Histone Methylation: Can either activate or repress transcription depending on the context and specific amino acids involved.

Epigenetic Inheritance and Tags

• Epigenetic tags can be passed on during cell division, maintaining specific gene expression patterns in differentiated cells of multicellular organisms.
• Reprogramming: Resetting epigenetic tags in gametes allows for tetrapotency, but can lead to loss of certain inherited traits.
• Imprinting: Differential expression of genes based on parental origin can affect offspring and contribute to developmental processes.

Environmental Effects on Gene Expression

• Air pollution that contains chemcials can lead to changes in gene expression related to immune responses, increasing disease suscepitbility.

Consequences of Epigenetic Tag Removal

• Imprinting: Results in monoallelic expression of certain genes, influencing traits in diploid cells and potentially leading to developmental disorder such as Tigons vs. Ligers.
• Epigenetic Origins: Differences in size between tigons (lion father) and ligers (tiger father) may arise from epigenetic factors influencing growth-related genes differently in each hybrid.

Monozygotic Twin Studies

• Identical twins help isolate environmental factors affecting gene expression, as they share the same genetic makeup but may have different environmental exposures.

External Factors Affecting Gene Expression

• Lactose: The presence of lactose in an E. coli environment triggers the expression of genes for lactose usage.
• It coverts lactose into allolactose, which acts as an inducer.
• With no lactose, the repressor (LacI) prevents transcription of the genes (lacZ, lacY, and lacA).
• Genes are transcribed only when lactose is present, leading to the production of enzymes (β-galactosidase, permease, and transacetylase).

Role of Estrogen in Endometrium Development

• Oestrogen is produced by the ovaries and enters target cells, such as those in the endometrium, during the menstrual cycle.
• Estrogen binds to estrogen receptors (ER), which are transcription factors located in the cytoplasm or nucleus of target cells.
• Translocation and Dimerization: Upon binding, the oestrogen-receptor complex undergoes a conformational change, translocating to the nucleus and dimerizing (forming a complex with another ER).
• The activated estrogen receptor complex binds to specific DNA sequences that are located in the promoter regions of target genes.
• This leads to the transcription of genes that create endometrial growth and vascularization.

Role of Testosterone

• It diffuses through the plasma membrane of cell and creates a hormone receptor complex
• The hormone-receptor complex moves to the nucleus where it interacts with DNA sequences
• It will enhance or repress genes in the target genes