Central Dogma of the transfer of biological information
DNA-> RNA-> Nucleic acid sequence
must be translated into an amino acid sequence >Protein
Three nucleotides
= 1 codon
= 1 amino acid
The chemical structure of DNA
● DNA is a polymer of nucleotides
● Nucleotides are phosphorylated nucleosides
● Nucleosides comprise a ribose sugar and a nitrogen base
● There are four common nitrogen bases; two with purine ring structures (adenine and guanine) and two with pyrimidine ring
structures (thymine and cytosine)
● Nucleotides are attached by the deoxyribose sugar through phosphodiester bond
● The order or sequence of nucleotides in the DNA polymer is the code for information storage
2 DNA chains are held together by H bonds , 3 bonds between C-G and 2 between A -T. Forming a double helix. The phosphate on the 5’ and sugar on the 3’
Anti parallel complementary double helix
semi-conservative -preserves intact the two halves of the parental DNA molecule
Polarity on DNA
-polarity based on the phosphodiester backbone
● Positions on the base are indicated by a number
● Positions on the sugar are indicated by a number with a prime (‘)
●DNA strands have 5′ to 3′ polarity
conservative- produces 2 DNA helices one with old DNA and the other with all new DNA
Semi conservative - produces 2 DNA helices one strand with old and one strand with new
dispersive - produces 2 helices with alternating old and new
DNA Replication
- A new DNA strand is polymerized in the 5′ to 3′ direction, reading the parent strand in the 3′ to 5′ direction
● Leading strand, runs in 3’ to 5’ direction – DNA replication proceeds in continuous manner
●Lagging strand, runs in 5’ to 3’ direction – replication apparatus will jump a short distance and then copies backwards making small fragments called Okazaki fragments
● Helicase unwinds and unzips the DNA double helix cutting and re-closing of the DNA sugar-phosphate backbone
●Primase builds an RNA primer for the DNA polymerase to start connecting the phosphate to sugar of the incoming nucleotide
●on the lagging strand, primase will repeatedly work to start each Okazaki fragment
●another enzyme will remove RNA primer and replace it with complimentary DNA bases
●DNA polymerase brings in and free complementary nucleotides
●Ligase ties together the Okazaki fragments by form phosphodiester bonds
Enzymes involved in DNA Metabolism
DNA Polymerases
Helicases:
Primase
Methylases:
Deaminases
Ligases:
● DNA Polymerases: catalyze the formation of phosphodiester bonds to extend the3’ ends of existing DNA chains. They require a DNA template in order to determine which base to add
● Helicases: unwind and untangle double-stranded DNA
● Primase: synthesizes a short ribonucleic acid (RNA) to prime DNA synthesis
● Methylases: add methyl groups to nitrogen bases
● Deaminases: remove amine groups from nitrogen bases
● Ligases: catalyze formation of a single phosphodiester bond between two adjacent nucleic acid fragments. Seals gaps and join two shorter pieces of DNA into one longer piece
DNA-Metabolizing Enzymes
Nucleases
Exonucleases: remove bases from the ends of DNA strands
Endonucleases: cut DNA strands internally
Restriction endonucleases cut double-stranded DNA
Recombination
●mixture & assembly of new genetic combinations
● Gene cloning is a method of in vitro recombination
● Recombination occurs in vivo by crossing over and then random assortment of chromosomes
Gametes will merge to form new diploid individual.
Recombinants
● Molecule that holds the new combination of DNA sequences
● produced in eukaryotes through
sexual reproduction
● produced in prokaryotes by gene
transfer through conjugation, transduction, or transformation
Plasmids
Plasmids are used to move genes from cell to cell
● Required to perform recombinant DNA technology:
o Plasmid vectors: for carrying DNA
o Restriction enzymes: for cutting DNA
o DNA ligase: for “pasting” DNA
3 ways DNA can be transferred between bacterial cells
- Conjugation – DNA is transferred between bacteria through a tube between cells
- Transduction – DNA is accidentally moved from one bacterium to another by a virus
- Transformation – bacterium takes up a piece of DNA floating in the environment
Gene
●ordered sequence of nucleotides on a chromosome that encodes a specific functional product
- Genes contain nucleotide sequences
that will be transcribed and/or
translated into protein
DNA vs. RNA
Double-stranded VS Single-stranded
* Deoxyribose sugars VS Ribose sugars
* Contains thymine VS Contains uracil
* Lasts (almost) forever VS Short half-lives
* All normal cells contain exactly
the same DNA VS Different cell type can have different RNA profiles
* Essential & always present VS Made only when needed
Types of RNA
●Messenger RNA (mRNA): take message from DNA to ribosome
● Ribosomal RNA (rRNA): part of the ribosome
● Transfer RNA (tRNA): transports amino acid to ribosome for building proteins
RNA Transcription
Initiation
● RNA synthesis begins at the promoter that tells the RNA polymerase to sit on DNA and start transcribing. The promoter has DNA sequences that let RNA polymerase attach to DNA - forming a transcription bubble to start transcribing There are -25 and -10 prokaryotic and -25 eukaryotic promoters. Because they come that may nucleotides +1 before the initiation site. In eukaryotes there is a TATA box to help RNA polymerase bind to promoters
Eukaryotes have distal elements, proximal elements before the promoter sequence. Prokaryotes dont have distal
Elongation- when the RNA strand gets longer along on strand of DNA in 3-5 direction .
Termination - RNA polymerase will keep transcribing until there are signals to stop known as termination
mRNA Processing: polyA tail, Capping & Splicing
- mRNA carry a sequence of polyadenylic acid at the 3’ end, polyA tail that help prevent RNA degradation allowing the RNA to be taken from the nuclease to be translated into a protein by ribosomes in the cytoplasm
- Capping: 5’ end is capped with a nucleotide that has a methyl & phosphate group. Marking as self
- allows mRNA to exit nucleus
- Splicing: introns are cut out and exons are brought together
Types of Gene Expression
constitutive genes are always expressed. Tend to be vital for basic cell functions (often called housekeeping genes)
Inducible genes are normally off, but can be turned on when substrate is present. Common for catabolic genes
Epigenetics
● Study of how cells control gene activity. Epigenetic factors also control the expression of genes
- Transcriptional gene silencing (e.g. imprinting, x-inactivation)
* Methylation
* Histones affect accessibility of DNA to bind to RNA polymerase - Post-transcriptional gene silencing (e.g. RNA-induced silencing complex) by small interfering RNA siRNA that cleave target mRNA
- Post-translational protein-protein interactions
Protein Structure
- Primary: amino acid sequence
- Secondary: intra-chain folding
o ⍺-helices or β-pleated sheets - Tertiary: further folding
o Loss of which denature protein - Quaternary: protein-protein interaction
function
* Monomers to multimers
Amino Acids
- neutral at a specific pH which is their isoelectric point
*joined together by -C-C-N- linkages or peptide bonds to make proteins - Amino acid content (type and order) determines protein structure and function. Extracellular domains that are charged and glycosylated and intracellular domains that are hydrophilic
Examples of Protein Functions
● Enzymes
● Transport
● Storage
● Motility
● Structural
● Defense
● Regulatory
Conjugated Proteins
Lipoproteins – lipid
● Glycoproteins – carbohydrate
● Metalloproteins – metal atoms
● Non–amino acid portion – non-protein prosthetic group
Protein Translation
● Messenger RNA – template
● Ribosomes – peptidyl transferase
● Transfer RNA (tRNA) – transports amino acid to ribosome
* Adaptors
Charging tRNA
- Amino-acyl tRNA synthetases covalently attach amino acids to tRNA’s, matching side chains and anticodons
1. amino acid + ATP → aminoacyl-AMP + Ppi
2. aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
Protein Translation
- Initiation
* Small ribosomal subunit binds to IF-3 & to 5’ mRNA
* This guides the AUG codon (start codon)
to the proper place in the subunit
* IF-2 & tRNAmet joins the complex
* Large ribosomal subunit will bind - Elongation
* tRNA carrying the next amino acids binds to the growing peptide chain - Termination
* Nonsense codon, stop codon, will stop protein synthesis
* Stop codons: UAA, UAG, UGA
Proteins are translated from mRNA by peptidyl transferase activity in the ribosome, using tRNA molecules as adaptors
