Molecular Genetics

The Central Dogma

REPLICATION


DNA replication is the process by which parental DNA is copied, producing two complete daughter genomes. This process originates at the origin(s) of replication, where a replication bubble opens up as the helicase enzymes unwind parent DNA strands. Helicase further widens the replication bubble by unwinding at the replication forks, while topoisomerase and single-stranded binding proteins stabilize the process. Meanwhile, RNA primase lays down a primer upon which DNA polymerase III begins adding nucleotides. The leading strand is produced as a continuous strand, whereas the lagging strand is made as a series of small fragments called okazaki fragments. DNA polymerase I then replaces the RNA primer with DNA nucleotides and DNA ligase welds together the okazaki fragments, leading strand and primer, to form a complete daughter strand.

TRANSCRIPTION


Transcription is the DNA-directed synthesis of messenger RNA (mRNA) that can carry a genetic message to ribosomes for protein manufacture. RNA polymerase initiates transcription when all the necessary transcription factors bind to the gene’s promoter. As the RNA polymerase moves downstream, it joins together RNA nucleotides and the RNA strand elongates, detaching from the parental template strand and allowing the DNA to rebind. In eukaryotes, this process continues until the RNA polymerase transcribes the polyadenylation signal sequence and proteins cut the pre-mRNA free from the polymerase

RNA PROCESSING


Before mRNA can exit the nucleus and bind to a ribosome in the cytoplasm, it must be modified. First a 5’ cap and a poly-A tail are added to its 5’ and 3’ ends respectively, to protect the mature mRNA from degradation by hydrolytic enzymes in the cytoplasm. Next, noncoding segments of the primary transcript, called introns, are removed by spliceosomes. The exons, untranslated regions (UTR), 5’ cap and poly-A tail are then spliced together to produce the final mRNA, which can direct the synthesis of a particular polypeptide. Since different segments can be considered introns or exons, a single gene may be able to code for different polypeptides by alternative RNA splicing.

TRANSLATION


During translation, the genetic message carried by mRNA is translated into the appropriate polypeptide or RNA molecule. After the mature mRNA exits the nucleus through a nuclear pore, it binds to a small ribosomal subunit, initiator tRNA and large ribosomal subunit, forming the translation initiation complex. tRNA plays a vital role in the elongation of the polypeptide by transferring cytoplasmic amino acids to the growing polypeptide chain. The anticodon at one end of the tRNA binds to the complementary codon of the mRNA and the corresponding amino acid is covalently bonded to its other end by aminoacyl-tRNA synthetases. As the tRNA moves from the ribosomes A site to P site, its amino acid is added to the polypeptide chain. The tRNA then moves to the E site and exits the ribosome. This process continues until a stop codon is reached and a release factor binds to the mRNA and hydrolyzes the bond between the polypeptide and the tRNA.

Mutations

Point mutations - single base change

base pair substitution

Silent mutation

Missense mutation

Nonsense mutation

No amino acid change because of redundancy in code

1 amino acid changed

Amino acid changed to stop codon

Lead to sickle cell anemia

missense mutation --> change from hydrophilic amino acid to hydrophobic amino acid

Framshift Mutations - shift in the reading frame --> changes everything downstream

Insertions - adding bases

Deletions - losing bases

Cystic Fibrosis - primarily whites of European descent, normal allele codes for a membrane protein that transports Cl- across membrane, defective or absent channels limit transport of Cl- across membranes --> thicker and stickier mucus coats around cells, mucus builds up in pancreas, lungs, digestive tract --> causes bacterial infections

Deletion of amino acid leads to cystic fibrosis

Control of Prokaryotic Genes

If they have enough of a product, need to stop production

Why? Waste of energy to produce more

How? Stop production of enzymes for synthesis

If they find a new food source, need to use it quickly

Why? Metabolism, growth, reproduction

How? Start production of enzymes for digestion

Feedback Inhibition - product as an allosteric inhibitor of 1st enzyme in tryptophan pathway, but this is a wasteful production of enzymes

Gene regulation - instead of blocking enzyme function, block transcription of genes for all enzymes in tryptophan pathway --> saves energy by not wasting it on unnecessary protein synthesis

Turn genes OFF

If bacteria has enough tryptophan then it doesn't need to make enzymes used to build tryptophan

Turn genes ON

If bacteria encounters new sugar like lactose then it needs to start making enzymes to digest lactose

Operon --> genes grouped together w/ related functions

Promoter: RNA Polymerase binding site, single promoter controls transcription of all genes in operon, transcribed as one unit and single mRNA is made

Operator: DNA binding site of repressor protein

Repressor Protein - binds to DNA at operator site, blocking RNA polymerase, blocks transcription

Synthesis pathway model - when excess tryptophan is present it binds to tryp repressor protein and triggers repressor to bind to DNA --> blocks transcription ---> Tryptophan is an allosteric regulator of repressor protein

Repressible

Inducible

Digestive pathway model - when lactose is present, binds to lac repressor protein and triggers repressor to release DNA --> induces transcription --> conformational change in repressor protein --> needs to make lactose-digesting enzymes --> lactose is allosteric regulator of repressor protein

Usually functions in anabolic pathways, when end product is in excess, cell allocates resources to other uses

Usually functions in catabolic pathways, produce enzymes only when nutrient is available, cell avoids making proteins that have nothing to do, cell allocates resources to other uses

Control of Eukaryotic Genes

Multicellular, evolved to maintain constant internal conditions --> homeostasis --> must coordinate body as a whole rather than serve the needs of individual cells

Packing/Unpacking DNA

Transcription

mRNA Processing

mRNA Transport

Translation

Protein processing

Protein degradation

From double helix --> nucleosomes --> chromatin fiber --> looped domains --> chromosomes

Nucleosomes --> beads on a string, 1st level of DNA packing, histone proteins

Positively charged amino acids bind tightly to negatively charged DNA, degree of packing of DNA regulates transcription

Heterochromatin - highly packed --> no transcription --> genes turned off

Euchromatin - loosely packed --> genes turned on

DNA methylation - blocks transcription factors --> no transcription --> genes turned off --> attachment of methyl groups to cytosines nearly permanent inactivation of genes

Histone acetylation --> acetylation of histones unwinds DNA, loosely wrapped around histones, enables transcription --> genes turned ON --> attachment to histones conformational change in histone proteins --> transcription factors have easier access to genes

Control regions of DNA

Promoter: nearby control sequence of DNA, binding of RNA polymerase and transcription factors

Enhancer: Distant control sequences on DNA, binding of activator proteins enhances rate of transcription

Activator proteins: bind to enhancer sequence and block gene transcriptions

Alternative RNA processing --> variable processing of exons creates a family of proteins

Life span of mRNA determins amount of protein synthesis

RNA interference

Small interfering RNAs, short segments of RNA bind to mRNA create sections of double stranded DNA --> death tag for mRNA --> triggers degradation of mRNA --> causes gene silencing --> turns off gene --> no protein produced

Block initiation of translation stage, regulatory proteins attach to 5' end of RNA, prevent attachment of ribosomal subunits and initiator tRNA blocks translation of mRNA to protein

Folding, cleaving, adding sugar groups, targeting for transport

Ubiquitin death tag --> marks unwanted proteins with a label, labeled proteins are broken down by proteasomes

Proteasomes --> protein degrading machine, cell's waste disposer, recycling

Viruses

Obligate intracellular parasite
virus consists of: genetic info, protein coat

Since they cannot carry out their life cycle independently they are not alive

Herpes, Measles, Polio, Smallpox

Virulent phages - phages that only replicate via the lytic cycle

Lytic cycle

Temperate phages - phages that replicate using the lysogenic and lytic cycle

Lysogenic Cycle

Phage therapy: treatment of bacterial diseases using phages, phages are species specific to bacteria

Eukaryotic Viruses - DNA or RNA genomes, single or double stranded, many have a lipid envelope that surrounds protein coat

Influenza- RNA Virus

HIV - retrovirus

genome is RNA but virus has code for reverse transcriptase which makes a DNA copy of the genome which is then spliced into the host cell chromosome, RNA-DNA

Plant Viruses: Appear as blotchy pigment pattern

Viroids: disease causing RNA molecules in plants, do not code for protein

Prions: Disease causing protein molecules --> have no genetic material, cause misfolding of normal proteins in the brains of various animal species

Corepressor binds to repressor protein, activating it

Inducer binds and inactivates repressor protein

Transduction: move from one host to another, viruses can pick up pieces of the first host's DNA and carry it to the next cell to be infected

RNA viruses lack replication error checking mechanisms and thus have higher rates of mutations --> leads to rapid mutation of viruses and different types

Genetic Engineering

Recombinant DNA- DNA that has been artificially made from different sources, ex: human gene into E.coli bacterium

Restriction enzymes: used to cut strands of DNA at specific locations (called restriction sites)

Restriction fragments: Cut will result in a set of restriction fragments, which may have at least one single stranded end, called a sticky end. Sticky ends can form hydrogen bonds with complementary single stranded pieces of DNA --> sealed by ligase

  1. Identify and isolate the gene of interest and the cloning vector
  1. Cut both the gene of interest and the vector with the same restriction enzyme
  1. Join the two pieces of DNA with ligase
  1. Get the vector carrying the gene of interest into a host cell
  1. Select for cells that haven been transformed

Limited host range

Mutagens: things that cause mutations: radiation and chemicals