Microbial Genetics

•Chromosome: discrete cellular structure composed of a neatly packaged DNA molecule

•Eukaryotic chromosomes-DNA wound around histones-located in the nucleus-diploid (in pairs) or haploid (single)-linear appearance

•Prokaryotic chromosomes-DNA condensed into a packet by means of histone-like

proteins-single, circular chromosome

Chromosomes

Genes Related to Obesity in the Human Genome

• Notice it is single & circular

• Does E. coli have 1 or 2 alleles of each gene? How do you know?

• Humans were first thought to function with 100,000 genes and now the number has dropped to ~35,000 genes although this is still a hot topic in research

Map of E. coli’s ~5000 Genes

Genome

E. coli cell disrupted to release its DNA molecule.

• A gene is a segment of DNA that contains the necessary code to make a protein or RNA molecule

•Three categories of genes• structural genes:

code for proteins

• genes that code for RNA

machinery used in protein

production• regulatory genes:

control gene expression

Gene

Genetic Terms• Genotype

• an organism’s genetic makeup; its entire complement of DNA

• Phenotype• is the expression of the

genes: the proteins of the cell and the properties they confer on the organism.

• Size, shape, color, environment

•Nucleotide: basic unit of DNA structure

• phosphate• deoxyribose sugar• nitrogenous base

•Nucleotides covalently bond to each other in a sugar-phosphate linkage

The DNA Code

5′

3′5′

5′

3′

DT A

D

CG

DG C

G C

P

P

P

P

P

D

D

A T

P

D

P

DT A

D

CG

P

P

P

P

P

P

G C

A T

5′

3′

4′

2′

1′D

P

P

P

D

P

P

H

H

H

H

H

H

N

NN

N

N

O

O

N

N

H

H

H

HN

N

NN

O

D

D

D

D

D

D

D

D

O

OO

OO

O

O

O

O

OO

OO

Sugar

OHN–H

H–

CH3O

(a)

OH

SugarN–

H–N

Hydrogen bond

N–H

Nitrogenous Bases and Base Pairing• Pairing dictated by the

formation of hydrogen bonds between bases

• Complementary Base Pairing– if sequence of one strand known, sequence of other strand inferred

• Try it:

TAC GTA ACG

ATG CAT TGCHydrogen bond

Nature of the Double Helix- Antiparallel arrangement:

one side of the helix runs in the opposite direction of the other

- One side runs from 5’ to 3,’ and the other side runs 3’ to 5’

- This is a significant factor in DNA synthesis and protein production

DNA Replication

DNA DNA

DNA Replication• DNA replication involves

unwinding a DNA double helix and using each strand as a template for a new, complementary strand

• DNA polymerase and over a dozen other enzymes and proteins are required to successfully replicate a single strand of DNA

• DNA replication is semi-conservative since each new chromosome will have one “old” and one “new” strand

• When does this occur??

DNA Replication• What is needed to

replicate DNA:

1. Original DNA template 2. Nucleotides

• a pool of nucleotides is free floating in the cytoplasm

3. Enzymes• DNA polymerase, ligase

4. Energy • ATP

DNA Replication: Prokaryotes• Certain enzymes

unwind the DNA.• Then, DNA

polymerase can read the parent strand and attach a complementary nucleotide to the new strand of DNA.

• Nucleotides are free in the cytoplasm.

15

Transcription

DNA RNA

DNA vs. RNA

– Contains ribose rather than deoxyribose

– RNA is single stranded– There is no T in RNA.

Instead it is a U:• A:U in RNA

– Can assume secondary and tertiary levels of complexity, leading to specialized forms of RNA (tRNA and rRNA)

Transcription: RNA Synthesis• What you need to synthesize RNA:

1. Original DNA template: • chromosome with a promoter

site (DNA sequence indicating start site) and a terminator site

2. Nucleotides• G, C, A, U Uracil is substituted for

thymine3. Enzymes

• RNA polymerase 4. Energy

• ATP

Transcription•RNA polymerase: large, complex enzyme that

directs the conversion of DNA into RNA

•Template strand: only one strand of DNA that contains meaningful instructions for synthesis of a functioning polypeptide

TranscriptionMany types of RNA can be transcribed:

1. Messenger RNA (mRNA)• RNA molecule that serves as a

message of the protein to be produced

2. Transfer RNA(tRNA)• Transfers amino acids to

ribosome 3. Ribosomal RNA (rRNA)

• Forms the ribosome4. Regulatory RNA

• micro RNAs, anti-sense RNAs, riboswitches, small interfering RNAs

Transcription: Initiation

• RNA polymerase recognizes promoter region• RNA polymerase begins its transcription at a special

sequence called the initiator• As the DNA helix unwinds it moves down the DNA

synthesizing RNA molecule

Transcription: Elongation

• During elongation the mRNA is built, which proceeds in the 5’ to 3’direction (you do not need to know the direction of elongation for this class)

• The mRNA is assembled by the adding nucleotides that are complementary to the DNA template.

• As elongation continues, the part of DNA already transcribed is rewound into its original helical form.

Direction oftranscription

Early mRNAtranscript

Nucleotidepool

Transcription: Termination

At termination the polymerases recognize another code that signals the separation and release of the mRNA strand,or transcript.

Late mRNA transcript

Elongation

Practice Transcription

• DNA: GCGGTACGCATTAAGCGCCC

• RNA:

Translation

mRNA Protein

Translation• Decoding the “language” of nucleotides and

converting/translating that information into the “language” of proteins.

• The nucleic acid “language” is in the form of codons, groups of three mRNA nucleotides.

• The protein “language” is in the form of amino acids

• Translation occurs at the ribosome• The green mRNA strand is “threaded” through the ribosome.• The ribosome “reads” the mRNA strand codons with the help of the

genetic code and tRNA

Translation

tRNA• Decoder molecule which

serves as a link to translate the RNA language into protein language – One site of the tRNA has an

anticodon which complements the codon of mRNA

– The other site of the tRNA has an amino acid attachment site corresponding to a specific amino acid as noted in the genetic code

Translation and the “Genetic Code” • Triplet code that specifies a

given amino acid

• We use the “genetic code” (at right) to translate mRNA nucleotide sequence (codons) into amino acid sequence which make up proteins.

• The “genetic code” is degenerate which allows for a certain amount of mutation. I.e. UUU and UUC both code for Phe

• There is one start codon, AUG, that codes for the amino acid methionine.

• There are 3 stop codons, UAA, UAG and UGA that signal the ribosome to stop translation and let go of the polypeptide chain (protein).

Translation and the “Genetic Code”

Practice Translation

• RNA:CGCCAUGCGUAAUUCGCGGG

1st Step: Find the start of the gene which is always indicated by AUG. Everything upstream from that can be ignored.

Practice Translation

• RNA:CGCCAUGCGUAAUUCGCGGG

1st Step: Find the start of the gene which is always indicated by AUG. Everything upstream from that can be ignored.

Practice Translation

• RNA:AUG/CGU/AAU/UCG/CGG/G

2nd Step: To make it easier to track the codons I separate each with a slash

Practice Translation

• RNA:AUG/CGU/AAU/UCG/CGG/G

3rd Step: Use genetic code to translate mRNA message into amino acid language

Translation at the Molecular Level: Initiation

• Ribosomes bind mRNA near the start codon (ex. AUG)

• tRNA anticodon with attached amino acid binds to the start codon

• Ribosomes move to the next codon, allowing a new tRNA to bind and add another amino acid

36

Translation at the Molecular Level: Elongation

• Two amino acids form peptide bonds

37

Translation at the Molecular Level: Elongation

• Stop codon terminates translation

38

Translation at the Molecular Level: Termination

Videos

https://www.youtube.com/watch?v=41_Ne5mS2ls

https://www.youtube.com/watch?v=5bLEDd-PSTQ&list=PL1AD35ADA1E93EB6F&index=2

Polyribosomal Complex

-A single mRNA is long enough to be fed through more than one ribosome

-Permits the synthesis of hundreds of protein molecules from the same mRNA transcript

-Would you see this in Eukaryotes?

Transcription and Translation in Eukaryotes and Prokaryotes

• Similar to prokaryotes except– AUG encodes for a

different form of methionine

– Transcription and translation are not simultaneous in eukaryotes

– Eukaryotes must splice out introns to achieve a mature mRNA strand ready to go to the ribosome.

-Only found in bacteria-Coordinated set of genes to make proteins

that are needed at the same time-all regulated as a single unit-either inducible or repressible

Operons and Gene Regulation

lac Operon• Most studied operon• When lactose is absent

the repressor blocks RNA Polymerase from binding to the operator and transcribing downstream genes.

• When lactose is present it binds to the repressor and it falls off the operator allowing RNA Polymerase to bind.

• The downstream genes are responsible for digesting lactose and are only on when lactose is present.

Phase Variation

• Bacteria turn on or off a complement of genes that leads to obvious phenotypic changes

• New environment new phenotype!• Most often traits affecting the bacterial cell

surface

• Examples: - Neisseria gonorrhoeae: production of attachment

fimbriae- Streptococcus pneumoniae: production of a capsule

Mutations• A change in the sequence

of DNA• Possible effects of

mutations• No effect-->no change in a.a.

sequence • Good-->new aa. Seq

– Increases variability in the gene pool, this is evolution!

• Bad-->new aa. Seq

• Cancer can be the product of a combination of bad mutations.

Types of Mutations• Point Mutation

• put the cat out--->puc the cat out• put the cat out--->put

• Frameshift (reading frame of mRNA shifts)• put the cat out--->put hec ato ut• Deletion• Addition• Duplication

The Effects of a Point Mutation

• When a base is substituted in DNA the mutation may have 2 effects:– Changes the amino acid– Does not change the amino

acid– Why doesn’t a mutation

always change the amino acid sequence?

The Effects of Frameshift Mutations

• The addition, deletion or insertion of one or more nucleotides drastically changes the amino acid sequence.

Mutation Rates• Normal Mutation Rate- 1/1 million per gene

– Mutations are constantly occurring since our enzymes are not 100% perfect.

• Mutagen- chemical or radiation that bring about mutations.

• Mutagen Mutation Rate= 1/1000-1/100,000 per gene (10-1000X the normal rate)

Mutagen Examples • 5-Bromouracil and acridine

are 2 mutagen examples that can “insert” themselves in DNA and cause errors in DNA replication, transcription and translation.

• Notice how similar in structure mutagens can be. There is just one change to thymine that can have dire consequences

Thymine Dimers Caused by Radiation

• Radiation, such as X-rays and UV rays, can cause dimers to form in DNA.

• Thymine dimers can interfere with DNA replication, transcription and translation.

What is the connection to cancer?• Cancer is a genetic disease. It is

the consequence of a change in DNA sequence.

• Carcinogen=substance that causes cancer

• Are mutagens also carcinogens? Yes

• Are all carcinogens also mutagens? No, alcohol and estrogen are carcinogens that speed up mitosis but do not directly cause mutations.

Ames TestThe Ames Test uses bacteria to identify possible carcinogens by looking for mutations to occur. Once a mutagen is identified, it is tested in animals to test if it is a carcinogen.

Genetic Recombination• During meiosis of human gametes• In bacteria, occurs when DNA is transferred between

bacteria.• Increases diversity in gene pool• End result is a new strain different from both the

donor and the original recipients• Vertical gene transfer-

• Genes/DNA passed from an organism to its offspring

• Horizontal gene transfer-• Genes/DNA transferred between organisms

-Depends on the fact that bacteria have plasmids and are adept at interchanging genes

-Provide genes for resistance to drugs and metabolic poisons, new nutritional and metabolic capabilities, and increased virulence and adaptation to the environment

Genetic Recombination

Plasmids• Self-replicating circular pieces of DNA• 1-5% the size of bacterial chromosome• “mini-chromosome”• Bacteria can store up many different plasmids for their use &

can transfer these to other bacteria. • They can contain any gene that the bacteria don’t require but

are useful to the survival of the bacteria. For example antibiotic resistance genes, toxin production, etc.

Antibiotic Resistance (R) Plasmids

• Some plasmids can carry many antibiotic resistance genes.

• When bacteria collect many plasmids and these plasmids have many antibiotic resistance genes, a “superbug” may originate.

Three Types of Genetic Transfer (Recombination) in Bacteria

ConjugationTransformation

Transduction

Conjugation• A donor cell contains a F

(fertility) plasmid making it F+.

• A conjugation pilus (genes for which are on the F+ plasmid) forms and the donor cell transfers a copy of the F plasmid to the recipient.

• Now, both cells have a F plasmid

• F+ plasmids can have other genes on them too, for example antibody resistance containing genes

Hfr Conjugation• High frequency recombination (Hfr) donors

contain the F factor in the chromosome• Donor gives part of its chromosome to the

recipient• This transfers more genes to the recipient

bacteria• Very fast evolution for the recipient!

Bridgebroken

Donatedgenes

Partial copyof donorchromosome

Integration ofF factor intochromosome

Pilus

DonorHfr cell

Transformation

• Occurs when naked DNA fragments of one bacteria are close to another living cell.

• Some bacteria have the ability to pick up naked DNA fragments and recombine the DNA into their own DNA

• The new recombinant cell now has some new DNA from the disintegrating cell. • The now transformed bacteria could have just picked up a new virulence factor or

antibody resistance

Griffith’s Classic Experiment to Test “Transformation Principle”

Mechanism of Transduction

• Virus mediated gene transfer

• The virus injects its genetic material into the bacteria

• The bacterial DNA is fragmented

Mechanism of Transduction• Viral particles are produced by

the bacteria• When the cell lyses, the viral

particles which have picked up DNA from the original bacterial cell now insert that DNA into a new cell.

• The new cell may or may not insert the new DNA sequence into its chromosome.

• Transduction can be a problem when the inserted DNA codes for an antibiotic resistance gene.

Transformation and Transduction in Research

Electroporation

A way to get the genes you want to work with into bacteria. Used in all types of molecular genetics research

Transposons• Transposons-

• Small segments of DNA that can move (be transposed) from one region of a DNA molecule to another.

• “jumping genes”– Involved in

• Changes in traits such as colony morphology, pigmentation, and antigenic characteristics

• Replacement of damaged DNA• Intermicrobial transfer of drug resistance (in bacteria)

Genes & Evolution

• Genes are continually altered due to mutation, recombination, and transposition

• These changes increase genetic diversity of the gene pool and then through natural selection adventitious genes may be selected for to ensure survival in many different habitats.

• For pathogens that means they are more virulent!