Genetics

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

Vertical Gene transfer

Horizontal gene transfer

Chapter 7

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

Diagrammatic representation of DNA

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

DNA Replication

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

RNA synthesis

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-sitebrings the correct amino acid to add at the A-site

Translation: RNA to protein

• What is needed for the process?– mRNA– Ribosomes– Amino acids– tRNA

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

The reading frame determines the protein

The Genetic code

Translation

Both processes occur at the same time in bacteria (why not

in eukaryotic cells?)

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

Models for transcriptional regulation with repressors

Transcriptional regulation by activators

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

Lactose Operon

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

If E. coli is growing in a flask with glucose and lactose…

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

Chapter 8

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

Vertical Gene transfer

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)

Base-pair mutation: missense

Results of base-pair mutations

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

Frameshift mutation

Induced mutations: transpositionTransposons = 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: Chemical mutagens

• Nucleobase modifiers

• Intercalating agents• Base analogs

Nitrous acid as a chemical mutagen

Nucleoside analogs are mutagens

Intercalating agents

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

UV light as a mutagen

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 site3. enzymatic cleavage of DNA strand4. enzymatic degradation of region of

strand the includes the incorrect nucleotide

Repair: Mismatch

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

Photoreactivation

Excision Repair

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

Chromosome transfer

• Hfr cells: have F plasmid integrated into chromosome

• Hfr cells produce an F pilus• F plasmid DNA directs transfer• A small amount of regional

chromosomal DNA is also shared in the transfer