Molecular Biology

DNA

Molecular Structure of DNA

• Made of repeated subunits of nucleotides

• Each has a five-carbon sugar, a phosphate, and a nitrogenous base

• Pentose-shaped sugar in DNA: deoxyribose

• Nucleotides can have 4 different nitrogenous bases

  • Adenine: a purine (double-ringed)

  • Guanine: a purine (double-ringed)

  • Cytosine: a pyrimidine (single-ringed)

  • Thymine: a pyrimidine (single-rined)

• Nucleotides linked together by phosphodiester bonds between the sugars and phosphates

  • Sugar-phosphate backbone of DNA

• 2 DNA strands*

• Each DNA strand wrap around each other to form twisted ladder, double helix

  • Deduced in 1953 by Watson, Crick, and Franklin

• A-T (2) and G-C (3) is known as base pairing

• Two strands are always complementary

  • If one side is ATC, then other is TAG

• DNA strands run ANTIPARALLEL

  • 3’ to 5’

  • The 5’ has the phosphate group and the 3’ has an OH or hydroxyl group

• DNA

  strands linked by hydrogen bonds (2 hold together adenine and thymine together and 3 hydrogen bonds hold cytosine and guanine together)

Genome Structure

• Each combination of the nucleotides is a gene (human genome has 20,000 genes)

  • The instructions of all the genes are spread among the nucleotides of DNA and all the DNA for a species is called its genome

  • Each separate chunk of DNA in a genome is called a chromosome

• Prokaryotes have one circular chromosome and eukaryotes have linear chromosomes (DNA more structured)

• DNA is wrapped around proteins called histones, and then histones are bunched together in groups of nucleosome

  • Chromosomes consist of DNA wrapped around proteins called histones

  • When the genetic material is in its loose form in the nucleus it is called euchromatin, with its genes active/available for transcription

  • When genetic material is fully condensed into coils: heterochromatin and its genes are inactive (DNA METHYLATION AND HISTONE ACETYLATION)

DNA replication

• DNA REPLICATION IS SEMICONSERVATIVE (ONE DNA MOLECULE CONTAINING 1 ORIGINAL STRAND AND A NEWLY SYNTHESIZED COMPLIMENT)

  • BUILDS 5-3 (reads from 3-5)

• Copying of DNA: DNA replication

• DNA molecule is twisted over itself and the first step is to __unwind the double helix by breaking hydrogen bond__s BY THE HELICASE which exposes DNA strands to form the replication fork

• Each strand serves as a template for the synthesis as another strand

  • DNA replication begins at specific sites: origins of replication

• Topoisomerases cuts and rejoins the helix to prevent tangling

• DNA polymerase: the enzyme that performs the addition of nucleotides long with the naked strand

  • Can only add nucleotides to the 3’ end of an existing strand

  • To start replication at the 5’, RNA primase adds a short strand of RNA nucleotides called the RNA primer (primer is later degraded by enzymes, and the space is filled with DNA)

• Leading strand: is made continuously (nucleotides steadily added one after another by DNA polymerase

  

• Lagging strand: made discontinuously in pieces known as okazaki fragments

• NUCLEOTIDES ARE BUILT IN 5’ TO 3’ DIRECTION (ADDED TO THE 3’ STRAND TO 5’ OF ORIGINAL)

  • Okazaki fragments eventually linked by DNA ligase to produce continuous strand

• When DNA is replaced, each new molecule has half the original molecule = semi-conservative

  • Helicase: unwinds double helix into 2 strands

  • Polymerase: adds nucleotides to an existing strand

  • Ligase: brings together the okazaki fragments

  • Topoisomerase: cuts and rejoins the helix

  • RNA primase: catalyzes the synthesis of RNA primers

Central Dogma

• DNA’s main role is directing the manufacture of molecules that work in the body

• DNA > (transcription in nucleus) > RNA > (translation in cytoplasm) > protein

RNA

• Messenger RNA (mRNA): temporary RNA version of DNA recipe that gets sent to the ribosome

• Ribosomal RNA (rRNA): produced in the nucleolus, __makes up part of the ribosom__e

• Transfer RNA (tRNA): shuttles amino acids to the ribosomes and is responsible for bringing the appropriate amino acids into place at the appropriate time by reading the message carried by the mRNA

• Interfering RNA (RNAi): small snippets of RNA that are naturally made in body or intentionally created by humans;

  • Can bind to specific sequences of RNA and mark them for destruction

Transcription

• Making an RNA copy of specific section of DNA code

• 3 steps: initiation, elongation, and termination

  • Unwind and unzip the DNA strands using helicase beginning at special sequences called promoters (docking site)

  • RNA is single-stranded so only one of the 2 DNA strands has to be copied

    • The strand that serves as template is known as the antisense strand/non-coding strand/template strand

    • The other strand that lies dormant is the sense strand/coding strand

• RNA polymerase builds RNA like DNA polymerase, only adding nucleotides to the 3’ side (5’-3’)

• Means that the RNA polymerase must bind to the 3’ end of the template strands

• When transcription begins, RNA polymerase travels along and builds an RNA that is complementary to the template strand of DNA- with replaced nucleotide base

  • Once mRNA finishes adding nucleotides and reaches a termination sequence, it separates from the DNA strand, competing transcription

RNA processing

• Prokaryotes: mRNA is complete

• Eukaryotes: the RNA must be processed before leaving the nucleus

  • The freshly transcribed RNA is called hnRNA (heterogenous nuclear RNA) and contains both coding and noncoding regions

  • Regions that express the code that will turn into proteins: exons

  • The noncoding regions in the mRNA are introns

• Splicing: the introns removed before the mRNA leaves the nucleus and is accomplished by an RNA-protein complex called a spliceosome

• poly(a) tail is added to the 3’ and a 5’ GTP cap is added to the 5’ end

  • Processes produce a final mRNA that is shorter than the transcribes data

Translation

• Turning mRNA to protein

  • Protein made of amino acids

  • The order of the mRNA nucleotides will be read in the ribosome in groups of three

• Three nucleotides are codons

  • Codon corresponds to a particular amino acid

• mRNA attaches to a ribosome to initiate translation and waits for the appropriate amino acids to come to the ribosome

  • tRNA comes and has a molecule that has a unique 3D structure that resembles a four-leaf clover

• One end of the tRNA carries an amino acid

  • other end called ANTICODON has 3 nitrogenous bases that can complementary base pair with the codon in the mRNA

  • tRNAs are between in protein synthesis and becomes charged/enzymatically attaches to an amino acid in the cell’s cytoplasm and shuttles to the ribosome

    • The charging enzyme involved in forming the bond between the amino acid and the tRNA require ATP

Translation: initiation, elongation, termination

• Ribosome attaches to the mRNA, Ribosome holds everything in place while the tRNAs assist in assembling polypeptides

Initiation

• 3 binding sites: A site, P site, E site

  • mRNA shuffles through from A>P> E and as mRNA codons are read, the polypeptide will be built

• The start codon for the initiation of protein synthesis is AUG which codes for methionine

  • Complementary anticodon UAC on the tRNA is the shuttle, when the AUG is read on the mRNA, it delivers the methionine to the ribosome

Elongation

• Addition of amino acids

• mRNA contain many codons and as each amino acid is brought to the  mRNA, it is linked it it neighboring amino acid by a peptide bond>> polypeptide

Termination:

• The synthesis of a polypeptide is ended by stop codons; when the ribosome runs into ⅓ stop codons

REVIEW:

• Transcription: mRNA is created from a particular gene segment of DNA

• In eukaryotes, the mRNA is processes by having its introns (noncoding sequences) removed

• To be translated, mRNA proceeds to the ribosomes

• Free-floating amino acids are picked up by the tRNA and shuttled over to the ribosome where mRNA waits

• Translation: the anticodon of a tRNA molecule carrying the appropriate amino acid base pairs with the codon on the mRNA

• As new tRNA molecules match up to new codons, the ribosome holds them in place, allowing peptide bonds to form between the amino acids

• The newly formed polypeptide grows until a stop codon is reached

• The polypeptide/protein is released into the cell

Gene Regulation (gene transcription and expression)

• Gene regulation can occur at different times (largest is before transcription: pre-transcriptional regulation; but can also occur post-transcriptional or post-translationally)

• Start of transcription requires the DNA to be unwound + RNA polymerase to bind at the promoter

  • Transcription factors can encourage/inhibit this from happening by making it easier/more difficult for the RNA polymerase to bind/move to the start site

  • Structural genes: genes that code for enzymes needed in a chemical reaction. These genes will be transcribed at the same time to produce particular enzymes

  • Promoter gene: the region where the RNA polymerase binds to begin transcription

  • Operator: a region that controls whether transcription will occur and is where the repressor binds

  • Regulatory gene: codes for specific regulatory protein called the repressor. The repressor is capable of attaching to the operator and blocking transcription. If the repressor binds to the operator, transcription will NOT occur. If the repressor does not bind to the operator, RNA polymerase moves along the operator and transcription occurs.

    

• Operon:  the region of bacterial DNA that regulated gene expression

• Example: Lac Operon (controlling the expression of enzymes that break down lactose)

  • INDUCIBLE: OFF UNTIL SWITCHED ON

  • Structural genes: three enzymes (beta galactosidase, galactose permease, and thiogalactoside transacetylase) are involved in digesting lactose coded for

  • Regulatory gene: the inducer, lactose binds to the repressor, causing it to fall off the operator and “turns on” transcription

• Example: trp operon

  • REPRESSIBLE: ON UNTIL SWITCHED OFF

  • Turned “off” in the presence of high levels of amino acid, tryptophan

  • When tryptophan combines with the trp repressor protein, it causes the repressor to bind to the operator that turns the operon OFF, blocking transcription

    

• Post-transcriptional regulation occurs when the cell creates and RNA but then decides that it should not be translated into a protein__; RNAi molecules can bind to an RNA via complementary base bearing that creates a double-stranded RNA- when it is formed, it signals to the destruction machinery that the RNA should be destroyed

  • Prevents it from being translated

• Post-translational regulation occurs when a cell has made protein, but doesnt need to use the protein yet.

  • Common for enzymes b/c when cell needs them, it needs it ASAP; easier to make them ahead of time and then turn them off/on as needed

  • Can involve binding w other proteins, phosphorylation, pH, cleagahe, etc

Mutations

• Mutations: error in the genetic code

• Can occur b/c DNA is damaged and cannot be repaired b/c DNA damage is repaired incorrectly

  • Damage can caused by chemicals/radiation; mistake from DNA/RNA polymerase

  • DNA polymerase has proofreading abilities; RNA polymerase do not (DNA is temporary molecule and is not a problem)

  • DNA is passed on from cell to cell in somatic cells and from parent> offspring

Base Substitution

• Nonsense mutations: these cause the original codon to become a stop codon, which results in the early termination of protein synthesis

• Missense mutations: cause the original codon to be altered and produce a different amino acid

• Silent mutations: happen when a codon that codes for the same amino acid is created; no change in the corresponding protein sequence

Gene Rearrangements

1. Insertions and deletions result in the gain or loss of DNA or a gene

   1. Usually results in a frameshift mutation

2. Duplications can result in an extra copy of genes and are usually caused by unequal crossing during meiosis/chromosome rearrangements

   1. May result in new traits b/c 1 copy can maintain the gene’s original function and 1 copy may evolve a new function

3. Inversions can result when changes occur in the orientation of chromosomal regions

   1. May cause harmful effect if inversion involves a gene/important regulatory sequence

4. Translocations occur when a portion of 2 different chromosomes (or single in 2 dif places) breaks and rejoins in a way that causes the DNA sequence/gene to be lost, repeated, or interrupted

BIOTECHNOLOGY

Recombinant data

• Recombinant DNA is generated by combining DNA from multiple sources to create a unique DNA molecule NOT found in nature

  • Ex. introduction of eukaryotic gene of interest like insulin into a bacterium for production

  • Bacteria can be hijacked and put to work to create proteins

• Genetic engineering: Branch of technology that produces new organisms or products by transferring genes between cells

Polymerase Chain Reaction (PCR)

• Ability to make billions of identical copies of genes within a few hours

  • Process of DNA replication is slightly modified

  • In a small PCR tube, DNA, primers, DNA polymerase, and DNA nucleotides are mixed together

  • In a PCR machine, the tube is heated, cooled, warmed many times; when heated the hydrogen bonds break, separating the double-stranded DNA. when cooled, the primers bind to the sequence binding region of dna we want to copy

  • When warmed, the polymerase binds to the primers on each strand and adds nucleotides on each template strand

• After 1st cycle, there are 2 identical double-stranded DNA molecules (second= 4 segments)

Transformation

• Transformation: the process of giving bacteria foreign DNA

• Genes of interest placed into small circular DNA plasmid; with genes in a vector

  • plasmid vectors usually contain genes for antibiotic resistance an restriction sites

• plasmids/gene of interest are cut with same restriction enzyme, creating compatible sticky ends; when placed together the gene is interested into the plasmid creating recombinant DNA and transforms the bacteria

• Similar: transfection which is putting a plasmid into a eukaryotic cell rather than bacteria cell

Gel Electrophoresis

• DNA fragments can be separated according to their molecular weight using gel electrophoresis

  • DNA and RNA are negatively charged and migrate toward positive pole of the electrical field

• The smaller the fragments, the faster they move in the gel and further toward + side

  • Used for crime scenes, DNA finger-printing