Transcription & Translation

• Gene/ Gene expression regulation:

  Regulation of gene expression includes a wide range of mechanisms that are used by cells to ↑/↓ prod of specific gene products (protein/ RNA). 
• Eg of gene regulation:

  • Trigger developmental pathways & processes for cellular differentiation → creation of different cell types → prod of diff proteins w diff ultrastructures that suit their functions. 
  • Adapt to new food sources like enzymes involved in lactose metabolism are expressed by E. coli only when lactose present & glucose absent. Rate at which certain protein molecules are synthesised varies acc to demand. 
  • Pigments in fur of Himalayan rabbits regulated by ℃. In cold climates, they can exhibit black coloration. In extreme warm weather, may even develop light/ white toenails. 
• Some proteins are synthesised continuously at a fixed rate & the genes coding for such proteins are said to be constitutively expressed. 
• At the genomic level, eukaryotes can control whether or not a gene can be transcribed by a chromosomal structure.
• Heterochromatin = highly compacted DNA that winds more tightly around histones. Formation of heterochromatin → inactivation of genes cus it prevents the RNA polymerase & transcription factors from reaching the promoter region of the gene. Wo transcription initiation complex, transcription cannot begin, & the gene cannot be expressed.
• Euchromatin = less compacted DNA that winds less tightly around histones. Formation of it → active gene expression as it promotes access of RNA polymerase & transcription factors to promoter region of genes, hence allowing formation of transcription initiation complex.
• The DNA x2 helix is associated with proteins called histones. DNA molecules are (-) charged, histone proteins are (+) charged. Thus the DNA molecule is held around the histones by electrostatic interactions. Most of the DNA is wound around 8 histone proteins to form nucleosomes & remaining DNA, aka linker, joins adj nucleosomes. 
• DNA methylation:

  Addition of methyl groups to selected cytosine (C) nucleotides located in the CG-rich seq. Makes DNA wind around histones tighter (heterochromatin). ∴ DNA methylation prevents transcription by blocking binding of transcription factors, hence preventing formation of transcription initiation complex. DNA demethylation is js opp.
• Histone deacetylation:

  Remove acetyl groups → restores (+) charge to histones → tighter interaction btw histones & (-) charged DNA (heterochromatin)→ promoter region inaccessible to RNA pol & gTFs → no transcription initiation complex. Histone acetylation is opp
• Enhancers & silencers, like promoters, are non-coding DNA seq. When activator proteins bind to enhancers, there will be an ↑ rate of transcription. When repressor proteins bind to silencers, there will be a ↓ rate of transcription.
• Post-Transcriptional:

  1. RNA splicing

  mRNA formed after transcription can be spliced in diff ways (i.e. alternative splicing), meaning 1 gene can generate diff mature mRNA, each having diff combos of exons, meaninngggg diff polypeptides will be formed through translation. 
• Translational:

  1. mRNA stability

  Longer half-life of mRNA transcript. The longer the mRNA remains in the cytoplasm 4 translation, the more protein product that can be formed.
• Post - Translational

  1. Covalent modifications
  2. Phosphorylation / dephosphorylation
  3. Protein degradation
• Further processing by cleavage and/or covalent modification such as glycosylation, disulfide bond formation, & attachment of prosthetic groups is required.  Mods occur when polypeptides pass through rER and Golgi apparatus. Proteins which are no longer needed will be degraded immediately.
• Non-coding DNA

  Any part of a genome that does not code for proteins/ RNA products (e.g. rRNA, tRNA). Plays important roles in the eukaryotic cell (e.g. regulatory functions). Also, non-coding DNA may have undiscovered functions that are possibly also important to explain their presence in genomes over hundreds of generations.

  Non-coding DNA forms a large component of eukaryotic genome. Size of a genome (i.e. amount of DNA) of an organism is not always proportional to its complexity (i.e. number of genes).
• Repetitive DNA sequences

  Largest component in non-coding DNA. Repetitive sequences can be found grouped together in tandem repeats which consists of a short nucleotide seq repeated consecutively. 
• Tandem repeats are used for DNA profiling which is a technique by which individuals can be identified & compared via their respective DNA profiles. The ttl DNA seq of a human is about genes (coding DNA) & non-coding DNA sequences. Hence, to distinguish human individuals, we don’t study the coding regions! We study the non-coding seq where unique differences can be found.
• Non-coding seq do not code for any protein/ RNA product & ∴ mutations occurring here are tolerated & allowed to accumulate over time.  
• Short tandem repeats (STRs) (2-6 bp seq repeated 15 - 100 x). Variable number tandem repeats (VNTRs) (20 - 100 bp seq repeated up to 1000s of times). DNA analyses showed that a non-coding DNA seq at a particular locus on a chromosome ≈ show small nucleotide seq differences in diff individuals. These differences aka DNA polymorphisms. In general, DNA polymorphisms at a particular chromosomal locus differ either in nucleotide seq or in numbers of tandemly repeated nucleotide units.
• DNA profiling is ≈ used in criminal investigations (forensics) & to settle paternity disputes. Forensic investigation: Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur. Paternity testing:  Children inherit one set of their chromosomes from each parent and thus should possess a combination of parental DNA and all DNA fragments produced by child should be produced by either the mother/ father
• Telomeres:

  Nucleotide sequences found at both ends of eukaryotic chromosomes. Telomeres are non-coding regions of DNA made up of a series of tandem repeat seq where each repeat is about 5-10 nucleotides long.

  Telomeres ensure genes are not lost with each round of DNA replication due to the end replication problem to prevent loss of vital genetic information. Telomeres protect and stabilise the terminal ends of chromosomes.
• Genes: 

  A gene is a heritable factor that influences a specific characteristic. It is a specific seq of nucleotides in a DNA molecule which codes for a specific seq of amino acids in a polypeptide chain. Along the length of the DNA molecule, at specific locations, there are diff genes each coding for a diff gene product. 
• Transcription happens in cytoplasm cus DNA molecules are too large to move through nuclear pores in nuclear envelope. To overcome this, part of the DNA info is transcribed into smaller mRNA molecules, which can pass through the nuclear envelope. 
• Transcription:

  The process in which the nucleotide seq of a gene (DNA) is used as a template to direct the synthesis of RNA (namely mRNA, tRNA and rRNA) made up of complementary base sequences. 
• Promoters are DNA seq which serve as a recognition site for binding of RNA pol & gTF to initiate transcription. Promoters are classified as non-coding DNA as they do not code for proteins/ RNA products. Only 1 of the 2 strands of the DNA, serves as a template for transcription, to direct synthesis of the RNA. This strand is known as the template strand. mRNA sequence transcribed will be complementary to the sequence of template strand 
• Within the gene, there are regions called

  • Exons
  • coding seq of a gene (codes for products like polypeptide/ rRNA/ tRNA)
  • Introns
  • non-coding seq found btw exons
  • needs to be excised before translation of mRNA
• Splicing of RNA in eukaryotes refers to the excising of introns & the joining of exons. It ↑ the no of diff mRNA & hence diff proteins an organism can produce w js 1 gene.
• Messenger RNA (mRNA)

  • Exists in a single-stranded form.
  • In prokaryotes, mRNA doesn’t need to be mod & can be used immediately for translation.
  • In eukaryotes, mRNA first undergoes splicing b4 it is transported to cytosol.
  • mRNA acts as a template for translation.
  • Each codon (read from 5’ to 3’) within the coding region of the mRNA represents an amino acid in a polypeptide. Seq of codons on mRNA will determine seq of amino acids in corresponding polypeptide chain. 
• Codon is a set of 3 DNA nucleotides or a set of 3 RNA nucleotides.
• Transfer RNA (tRNA)

  • Exists in a single-stranded form but folds back upon itself & is held in shape by H bonding btw complementary base pairs at certain regions to form a 3D conformation/shape.
  • Has 3 loops.
  • On one of the loops, 3 specific triplet bases form an anticodon that binds to a specific mRNA codon via complementary base-pairing.
  • 3’ end is the attachment site for a specific amino acid that corresponds to the anticodon.
• tRNAs bring in specific amino acids in a seq corresponding to the seq of codon in mRNA to the growing polypeptide.

  
  

  It can facilitate translation due to:

  • Its ability to bind to a specific single amino acid.
  • The ability of its anticodon to base-pair with the mRNA codon.
• Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes
• Ribosomes are organelles that synthesize polypeptides using mRNA as the template, serving as the site for translation
• A ribosome starts translation at the 5’ end of mRNA and moves towards the 3’ end
• rER-bound ribosomes synthesize proteins for secretion or for use in lysosomes, while free ribosomes synthesize proteins for internal use within the cell
• The small subunit of a ribosome has an mRNA binding site
• The large subunit of a ribosome has 3 binding sites:

  • Aminoacyl site (A site) holds the incoming tRNA carrying the next amino acid to be added
  • Peptidyl site (P site) holds the tRNA carrying the growing polypeptide chain
  • Exit site (E site) is where the tRNA leaves the ribosome
• All proteins are made up of the permutations of 20 diff amino acids. The same triplet of nucleotides codes for the same amino acid in all organisms (universal code). For some amino acids, the same amino acid may be coded for by several codons (degenerate code). 3 nucleotides code for 1 amino acid. For eg: this short mRNA, AUGCCCGGAGGC, seq of amino acids coded by it is Met-Pro-Gly-Gly. ≥ 20 diff tRNAs (each with unique/specific anticodons) should exist in any cell.
• Translation is a process by which the seq of ribonucleotides in an mRNA molecule is converted into a seq of amino acids in a polypeptide chain. B4 translation can occur, each tRNA must be attached to the correct amino acid, a process aka amino acid activation. 
• Amino Acid Activation Prior to Translation:

  Involves:

  • Amino acid
  • ATP (used to form covalent bond btw amino acid & tRNA)
  • tRNA activating enzyme (Active site fits: a specific amino acid, a matching tRNA & ATP
  • tRNA which carries the specific amino acid that corresponds to its anticodon = activated amino acid