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What is genetics?
• The science of heredity; includes the study of genes, how they carry information, how they are replicated, how they are expressed
Adaptation and Natural Selection
• How do organisms adapt to change?
– Two basic options: regulate gene expression or change the genetic code
– Change in genetic code = mutation
Why use bacteria to study mutations?
• Only have one chromosome…one copy of each gene
• Easy to grow
Direct selection
• Testing for traits that are easily identified– Colony color– Motility– Resistance to antibiotics
Indirect selection
• A way to look at traits that are not easily identified, at changes in metabolic pathways
• Replica plating– A way to identify AUXOTROPHS from
PROTOTROPHS
What do you know about DNA?
• Chromosomes made of DNA make up an organism’s genome
• DNA codes for genes = functional unit of the genome
• Genes code for proteins• Chemical composition =
nucleotides
Replication: duplication of the genome prior to cell division
Gene expression: decoding of DNA in order to synthesize gene products (proteins):
Transcription: DNA →RNATranslation: RNA → protein
DNA Structure
• Double helix formed by complementary strands
• Strands composed of deoxyribonucleotide subunits = nucleotides
• Antiparallel strands held together by hydrogen bonds between base pairs– 5’ P04 binds to 3’ OH– Thymine pairs with adenine– Guanine pairs with
cytosine
Enzymes necessary for DNA replication
• Primase: synthesizes the RNA primer• Helicase: “unzips” 2 strands of DNA• DNA Polymerase: synthesize 5’→3’• DNA gyrase: releases tension during
uncoiling of circular DNA– Produced by prokaryotes and some simple eukaryotic
organisms only, so potential target for antibiotics
**target of quinolones and aminocoumarins**
• DNA ligase: seals the gaps between Okazaki fragments (forms covalent bonds)
Gene Expression
• Transcription• Post-transcriptional modification• Translation• Post-translational modification
Transcription: DNA to RNA
• RNA polymerase– Does not require a primer to initiate
synthesis– Recognition of the promoter via sigma
factor (bacterial transcription factor)• Process begins at the promoter region
and ends at the terminator sequence• Process proceeds in the direction 5’→3’• Base pairing: thymine replaced with
uracil; U-A, G-C
What are the possible products from transcription?
• Messenger RNA (mRNA)• Transfer RNA (tRNA)• Ribosomal RNA (rRNA)
Translation: RNA to protein
• What is needed for the process?– mRNA: has the code– Ribosomes: present the codons to tRNA,
align the amino acids • Protein + rRNA
– Amino acids– tRNA: anticodon ; initiates the
protein sythesis at the P-site
brings the correct amino acid to
add at the A-site
Initiation of Translation• Ribosome binds ribosome binding site
– on mRNA molecule– In bacteria: binding occurs during mRNA
synthesis – so translation and transcription occur simultaneously
• Ribosome completes assembly while bound to the mRNA
• Initiating tRNA binds to start codon: AUG – N-formylmethionine = f-Met)– Also codon for normal methionine
Elongation of the Polypeptide Chain
• 2 binding sites on ribosome for tRNA:– P-site:– A-site:
• Initiation tRNA binds to P-site and provides f-Met
• tRNA recognizing the next codon binds to A-site and provides coded AA
• Ribosomal enzyme creates a peptide bond between
Termination of Translation
• Ribosome gets to stop codon• No tRNA recognizes the stop codon
→enzymatic cleavage of bond that binds the polypeptide to the mRNA
• Ribosome falls off and dissociates into 2 subunits
• Subunits are ready to reassemble and initiate translation at another site
Post-Translational Modification
• Synthesized polypeptides are straight chains of amino acids
• Modifications to make them into functional proteins, ready them for transport out of the cell = PTMs
• Folding: chaperone-assisted• Tag removal: export signal sequence is
removed in the process of crossing the cytoplasmic membrane
Eukaryotic cells differ in transcription and translation
• Ribosomes are 80s – 40s and 60s subunits• 5’ end of mRNA is capped
– Methylated guanine added to pre-mRNA– Stabilizes transcript, enhances translation
• Polyadenylation of 3’ end of mRNA– Poly A tail added to pre-mRNA– Stabilizes transcript , enhances translation?
• Splicing: removal of non-coding sequences = introns; exons spliced together
• Translation is monocystronic
Is protein synthesis regulated?
• Three types of protein regulation– Enyme inhibition (ex: feedback inhibition)– Repression (ex: tryptophan operon)– Induction (ex: lactose operon)
Does regulation occur at the level of transcription?
• Some gene expression is constitutive: proteins encoded by these genes are continuously synthesized
• Other genes are induced: proteins only made when needed
• Other genes are repressed: proteins produced routinely, but turned off when not needed
Lactose Operon as a model
• Used to understand control of gene expression in bacteria
• Operon consists of three genes needed to degrade lactose
• Repressor gene (codes for repressor protein) outside of operon coding region inhibits transcription unless something else binds to the repressor protein
What conditions are needed for the lactose operon to be turned “on”?
• No glucose• Lactose present• Increasing levels of cAMP• cAMP binds to CAP, then complex binds
next to lactose operon promoter at the activator region
• RNA polymerase binds to promoter
Gene regulation systems in bacteria• Signal transduction:
transmission of information from outside to inside cell– Quorum sensing: ability
to sense the density of cells within the same population
– Communication occurs via molecular signals
– In quorum sensing, response to the signal is concentration dependent
– Critical level → induction of gene expression
Adaptation and Natural Selection
• How do bacteria adapt to change?• Like any organisms, they have 2 basic
options:– Regulate gene expression– Change the genetic code
• Change in genetic code = mutation• Bacteria can also utilize HORIZONTAL
GENE TRANSFER
What are mutations?
• Changes in the base sequence of the DNA
• Do they always change the genetic code?
What can cause mutations?• Chemicals (nitrous acid)• Physical mutagens (uv light)• Biological mutagens (transposons)• Spontaneous mutations (errors in
replication)– Random occurrences– Low frequency; usually at a constant within
a given population– Essential for a population to adapt to
change
Causes of mutations in bacteria
• Most are spontaneous– Errors made by DNA Polymerase
• UV light exposure• Oxidative injury induced by reactive
oxygen species (ROS) – superoxide, hydrogen peroxide
Types of Mutations• Base substitution: replacement of one
nucleotide base with another– Missense mutation: altered codon specifies a different
amino acid– Nonsense mutation: altered codon is a stop codon,
resulting in formation of a truncated, usually non-functional protein
– Silent mutation: the strict definition = a change in the codon does not change the encoded amino acid; a more broad definition = a change that does not change the function of the encoded protein• by this definition a silent mutation could be any of these types of
base substitions, as long as the function of the protein (phenotype) was not affected)
Types of Mutations
• Frameshift: deletion or addition of a nucleotide base– Changes the reading frame– Most result in a truncated, non-functional
protein = knockout mutation
Induced mutations: transposition
Transposons = segments of DNA that can move from one location in a cell’s genome to another
- Barbara McClintock: “jumping genes” biological mutagen- Most contain transcriptional terminators
Induced mutations: Radiation
• Ultraviolet light: introduction of thymine dimers– Covalent bonds form between adjacent thymine
molecules– Alters shape (distorts) double helix– Replication and transcription can’t proceed past
the site of distortion– SOS repair is initiated →increased risk of errors
• X rays: double and single strand breaks in DNA + nucleobase alterations
Repair mechanisms• Wrong nucleotide inserted
– Proofreading by DNA polymerase– Mismatch repair: fixes errors missed in
proofreading
1. recognition of mismatch (i.e., A-G)
* the non-methylated DNA strand is the new strand and therefore the one that is incorrect if a mismatch is present
2. protein binds to site
3. enzymatic cleavage of DNA strand
4. enzymatic degradation of region of strand the includes the incorrect nucleotide
Repair of UV damage• Two repair mechanisms
– Photoreactivation (light repair): • Enzymatic cleavage of covalent bonds between
thymine molecules• Uses energy from visible light to break the
bonds• Restores original DNA molecule
– Excision repair (dark repair):• Removal of strand of DNA containing thymine
dimers• DNA polymerize synthesizes replacement • DNA ligase binds the segments together
SOS Repair
• Last ditch effort: fix or die• DNA polymerase synthesized in
response to severe DNA damage does not proofread – quick and dirty transcription, error prone →
SOS mutagenesis
DNA-mediated Transformation
• Transduction– Specialized– Generalized
• Conjugation– Plasmid transfer– Chromosome transfer
Plasmid transfer
• Making contact: F pilus of donor binds to receptor on cell wall of recipient bacterium
• Initiation of transfer• Transfer of DNA• Transfer complete
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