microbial genetics: from genotype to phenotype nucleic acid synthesis protein synthesis heredity...
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Microbial Genetics:From Genotype to Phenotype
Nucleic acid synthesis
Protein Synthesis
Heredity
Genetic Recombination
Microevolution
Review Nitrogenous basedNTPBase pairingDNARNACoding strandTemplate strand
Notable differences in hereditary material in viruses, prokaryotes and eukaryotes
Important basis for modern methods of identification and disease diagnosis
Microbial Genetics
What does DNA do?Bacterial cell
DNA replication: Cell Division requires duplication of DNA
transcription
translationClones
RNA
DNA
protein
Large molecules of DNA are called chromosomes
Bacteria have circular chromosomes and sometimes additional small, circular pieces of DNA called plasmids
Replication of DNA
• Because DNA is double stranded replication can occur on each strand simultaneously in opposite directions
• Continuous, newly synthesized strand added in 5’ to 3’ is leading strand
• DNA synthesized from behind by the addition of Okazaki fragments is lagging strand, olignucleotides are still added to the 3’ end
DNA replication
Original double stranded DNA
Two new strands (green dotted lines) are created using a polymerase enzyme
The process of creating the new strands is called polymerization
DNA polymerization
3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…………….5’
5’ATCGAACGGAGACTTACTCTTA3’
DNA Polymerase
C A
A G
C
A
T
A
G
T
T
A
dNTPs are are added to the new strand (green) so that the dNTPs pair with the corresponding ones on the complimentary strand (black)
Sugar phosphate backbone Nitrogenous bases
DNA Polymerization
• Polymerization, requires DNA polymerase enzyme• Synthesis occurs only from 5’ to 3’ of new strand • dNTP incorporated into 3’ end of new strand by DNA
polymerase• Formula for polymerization:
(dNMP)nDNA +dNTP(dNMP)n+1DNA + PPi
• (dNMP)nDNA is the growing strand
• dNTP is the deoxynucleotide triphosphate
• (dNMP)n+1DNA is the growing strand after a dNTP is added
• iPP is inorganic diphosphate
Synthesis occurs on both strands of original DNA in opposite directions
Original strands are separated generating replication forks
5’
3’
Leading strandLeading strandLagging strands
Okazaki fragments
Nucleic acid polymerization
Nitrogenous base
Nitrogenous base
Nitrogenous base
Nitrogenous base
Nitrogenous base
Nitrogenous base
Nitrogenous base
Nucleotide triphosphate
5’
Polymerase enzyme
Inorganic phosphate
Transcription is synthesis of RNA from DNA
• NTPs (ATP, CTP, GTP, UTP) incorporated in to strand of mRNA (RNA polymerization)
• mRNA is complementary to the template strand of DNA• RNA polymerase enzyme is used
What is a gene?
DNA
rRNA
tRNA
mRNAprotein
mRNA synthesis occurs on the template strand (-) of original DNA
Original strands are separated where transcription is to begin
5’
3’
Synthesis of mRNA
Double stranded DNA has a coding strand (+) and template strand (-)
+
Synthesis of mRNA
3’TACCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…………….5’
5’AUGGAACGGAGACUUACUCUUA3’
RNA Polymerase
C A
A G
C
A
U
A
G
U
U
A
Template strand of DNA
AUG is start codon
Genetic Code for Building Proteins
Every three bases (triplet) of DNA corresponds to a codon of mRNA which corresponds to an anticodon in tRNA which bares a specific amino acid
Use the table in your book to provide the amino acids for the following DNA sequence on the coding strand :
CGTCCCGTC
Eukaryotes
E I E I E I E I E I E I E I E
E E E E E E E E
II I
II
I
I
DNA contains introns and exons
Exons have the code to build proteins
70S ribosome (prokaryotes)
30S subunit
50S subunit
21 different proteins
16SrRNA
23S rRNA and 5S rRNA
31 different proteins
Components of Prokaryote Ribosome
Ribosomes
The site of protein synthesis
Prokaryotes have 70S ribosomes
Eukaryotes have 80S ribosomes
Because ribosomes do such an important job drugs that inhibit them have drastic effects on the cell (many antibiotics and the toxin ricin inhibit protein synthesis by binding to ribosomes)
The genes for ribosomal RNA are often used to measure the similarity between organisms
16SrRNA gene for bacterial systematics
18SrRNA gene for eukaryote systematics
Translation
mRNA
Growing polypeptide connected by enzymatic linkage between amino acids
tRNA
ribosome
Example of regulation of protein synthesis: Enzyme Induction
Prom. Oper. Gene
mRNA
Pol.
repressor
Substrate binds to repressor allowing polymerase to transcribe mRNA
Enzyme synthesized
substrate
Enzyme acts on substrate
Example of regulation of protein synthesis: Enzyme Induction
Prom. Oper. Gene
Gene turned ‘off’Pol. repressor
In the absence of substrate, the repressor blocks polymerase enzyme
Note: the status of a gene being turned on or off is not heritable. While it allows individual organisms to adapt to changing circumstances, it does not effect the evolution of species as does mutation.
Mutation
• Point mutation -results from the replacement of a nitrogenous base effects may 1. be “silent” causing no change in the protein structure, or 2. result in altered protein with different performance or 3. produce protein that doesn’t work
• Frameshift mutation -results from the insertion or deletion of bases, has dramatic effects on protein, often deleterious.
the red fox ran out
the red fax ran out
thr edf oxr ano ut
After a point mutation the sentence might be:
After a frame shift mutation (deletion of letter e) the sentence might be:
The genetic code is analogous to written language
Suppose the sentence below is a genetic sequence:
Mutagens
• Chemicals that bind to DNA and affect the process of replication or transcription. Some naturally occurring chemicals are mutagenic, many industrial chemicals are mutagenic
• Radiation, including UV light, is mutagenic
Genetic Variation Results from Mutation
Most mutations are either harmful, or neutral, but sometimes they are beneficial.
If the mutations are not too harmful, they will be passed on to their progeny (offspring). This is the hereditary basis of evolution.
These heritable changes in a lineage or populations of organisms over generations contribute to micro-evolution
Antibiotic Resistance in Bacteria
• Bacteria can multiply rapidly into large populations that reach a stationary phase
• Antibiotic therapy or accidental ingestion of antibiotics acts as an agent for natural selection
• Single point mutation in DNA can lead to resistance in a single mutant bacterium
• Mutant ‘lost in crowd’ until antibiotic therapy kills off susceptible bacteria
• Mutant becomes dominant in population and gives rise to mutant clones
Variation by Mutation is Compounded by Genetic Recombination
• Sexual reproduction
• Bacterial transformation
• Bacterial conjugation
• Virus-mediated gene transfer
• Other transfer between symbionts
Sexual reproduction is genetic recombination
zygote
parent cells produced gametes by meiosis
gametes
Gametes fuse to form a zygote
2N diploid stage
1N haploid stage
egg sperm
gametogenesis fusion of gametes
Bacterial Transformation
Bacterium with gene for capsule
Bacterium without gene for capsule
Heat-killed bacterium disintegrate and release DNA into surroundings
Genes taken in and expressed
Conjugation
1. Plasmids may contain genes that allow a bridge (pilus) to form between two bacteria. Bacteria with these plasmids are F+
2. The plasmid from the F+ (donor) bacterium is replicated and transferred to the F- (recipient) bacterium
F+ F-
pilus
F+ F-
1. 2.
High frequency recombinations
F+
chromosome
Hfr+
Plasmid inserts into chromosome
Hfr+ recipient
recipient
Hfr+ DNA transferred into recipient and inserted into chromosome
Transposable elements (jumping genes)
• A mobile genetic sequence that can move from one plasmid sequence to another sequence or to a chromosome
• May result in the disruption of gene activity• Make up a large portion of Eukaryote DNA
Transduction
• Some viruses can incorporate their genetic material into the host’s chromosome
• When the virus is transferred from host to host, some of the host’s genes can be transferred along with the virus’s genes into the next host.
Bacterium with its chromosome containing viral (Prophage) DNA
Phage infects new bacterium bringing with it DNA from the previous one
Prophage DNA excised from host chromosome
Phage DNA inserts into new host chromosome along with bacterial genes
Transduction