Microbiology - Ch. 7: Microbial Genetics
DNA Replication Structure
- Complementary structure of the two strands - Replication is SEMICONSERVATIVE - new DNA composed of 1 original & 1 daughter strand - It is an Anabolic Polymerization Process that requires monomers & energy -- Deoxyribonucleotides Triphosphate serve both functions
Simplest Transposons
- Insertion sequences - Have no more than 2 inverted repeats and a gene for transposase
Transformation - Horizontal Gene Transfer - Prokaryotes
- One of conclusive pieces of proof that DNA is genetic material - Cells that take up DNA are competent -- results from alterations in cell wall and cytoplasmic membrane that allow DNA to enter cell
Methods to Recognize Mutants
- Positive Selection - Negative (indirect) Selection - Ames Test
Mutagens - Mutations of Genes
- Radiation Ionizing Radiation Nonionizing Radiation - Chemical Mutagens Nucleotide analogs Nucleotide-altering chemicals Frameshift mutagens
Transposons & Transposition
- Segments of DNA that move from one location to another in the same or different molecule - Result is a kind of frameshift insertion - transpositions - Transposons all contain PALINDROMIC SEQUENCES at each end
Bacterial DNA Replication
-4 DNA Polymerases running - 2 Leading & 2 Lagging -Bidirectional: 1 Origin of Replication, 2 Replication Forks -Gyrases & topioisomerases remove supercoils in DNA -DNA is METHYLATED at A & C residues: Control of genetic expression, Initiation of DNA replication, protection against viral infection -- no methylation on phage DNA, Repair of DNa - shows which was the original strand vs. newly synthesized strand
Prokaryotic Transcription - Termination
-At intrinsic terminators, a GC rich hairpin forms upstream of a poly U tract causing termination -Rho mediated termination: the Rho protein rides along the RNA 5' -- 3' until it catches the RNA polymerase then pulls the RNA out, causing termination
Bacterial Transcription - Initiation & Termination
-Bacterial promoters have sequences that are recognized by a sigma factor (RNA Pol subunit) -Intrinsic terminators have dyad semetry upstream of a poly U tract -Termination mediated by RNA hairpin
mRNA Processing Before Translation - Eukaryotes
-Capping: 5-methyl-guanosine cap on 5' end -Polyadenylation: AAAAAAAAAAAA on 3' end -Splicing: Removes introns, leaves exons (Exons are Expressed)
Mutations of Genes
-Change in the sequence of a genome -Rare event because they are dangerous -Almost always deleterious -Rarely leads to a protein that improves ability of an organism to survive
Bacterial Transcription - Elongation
-Genes can be 1000s of bases l ong, RNA polymerase has foot print of ~20 bp -Multiple RNA polymerases can be transcribing the same gene at the same time (often case in rRNA) -Prokaryotes - translation happens at the same time as transcription
Regulation of Genetic Expression
-Housekeeping genes are expressed at all times -Other genes transcribed & translated when cells need them - allows cell to conserve energy -Regulation of polypeptide synthesis: typically halts transcription; can stop translation directly
Inducible Operon - Prokaryotic Operons
-Must be activated by inducers Ex. Lactose operon
Prokaryotic Translation
-Occurs at the same time as transcription -Bacterial mRNAs can have multiple proteins encoded in them (polycistronic) -Begins at the Shein-Delgarno sequence -Initiation begins with a N-formylmethionine
Eukaryotic Translation
-Occurs outside the nucleus -mRNAs usually code a single gene (monocistronic) -Begins by binding the 5-methylguanosine cap of RNAs; scans START codon
Translation
-Process in which ribosomes use genetic information of nucleotide sequences to synthesize polypeptides -Mediated by tRNAs which read codons (3 base codes)
Eukaryotic Transcription
-RNA transcription occurs in nucleus -Transcription also occurs in mitochondria and chloroplasts -At least 3 types of RNA Polymerase (POL 1, 2, 3) -Numerous transcription factors -mRNA processed before translation
Frequency of Mutation
-Rare events -Mutagens increase the mutation rate by a factor of 10-1000 times
Control of Translation - Regulating Gene Expression
-Regulatory RNAs can regulate translation of polypeptides
Translation - Termination
-Release factors recognize STOP codons - modify ribosome to activate ribozyme -Ribosome dissociates into subunits -Polypeptides released at termination may function alone or together
Eukaryotic DNA Replication
-Similar to bacterial replication -Differences: Uses 4 DNA polymerases, thousands of replication origins, shorter okazaki fragments, plant & animal cells methylate only cytosine bases
Leading Strand in DNA Replication
-Synthesized CONTINUOUSLY -Uses only 1 RNA primer & proceeds in the same direction as replication fork
Lagging Strand in DNA Replication
-Synthesized DISCONTINUOUSLY -Proceeds in opposite direction as replication fork, then loops back to the fork -Each fragment is an OKAZAKI fragment -After initial synthesis, RNA primers are removed by and filled in with DNA by DNA Polymerase I and DNA Ligase
Repressible Operon - Prokaryotic Operons
-Transcribed continually until deactivated by repressors Ex. Tryptophan operon
Initial Process In DNA Replication
1) Bacterial DNA replication begins at ORIGIN 2) DNA Polymerase replicates DNA only 5' --> 3' 3) Because strands are antiparallel, new strands are synthesized differently -- Leading Strand & Lagging Strand (Bacteria- 1 Origin) (Eukaryotes- Many Origins)
Transduction - Horizontal Gene Transfer - Prokaryotes
1. Generalized Transduction: transducing phage carries random DNA segment from donor to recipient 2. Specialized Transduction: Only certain donor DNA sequences are transferred
Stages of Translation
1. Initiation 2. Elongation 3. Termination All stages require additional protein factors Initiation & Elongation require energy (GTP)
Prokaryotic Chromosome
A circular, double-stranded molecule of DNA together with a few attached proteins & RNA; Haploid (single chromosome copy)
Ionizing Radiation - Mutagens - Mutations of Genes
Alpha, Beta, Gamma radiation and cosmic rays
Termination in Transcription
Bacteria - happens at intrinsic terminators & by Rho mediated termination Eukaryotes- not well understood
Nucleosides
Base + Sugar -- No phosphate group
Prokaryotic Transcription - Initiation
Begins at promoters, mediated by SIGMA FACTOR which provides promoter specificity
Wild types
Cells normally found in nature
Genetic Recombinants
Cells with DNA molecules that contain new nucleotide sequences
Point Mutation - Mutations of Genes
Change consisting of one base - Substitution - Insertion - Deletion Insertions & Deletions cause frame shifts because the nucleotide triplets after the mutation are displaced forward or backwards one position
Plasmids
Circular DNA molecules that can replicate independently of the main chromosomes of bacteria; Origin of Replication; More than 1 copy/cell; NOT ESSENTIAL for metabolism, growth, reproduction; can confer survival advantages (resistance factors)
Complex Transposons
Contain 1 or more genes not connected with transposition
Transcription
DNA --> RNA 1st Step in Gene Expression
Central Dogma of Molecular Biology
DNA --> RNA --> Proteins DNA transcribed to RNA translated to Protein
Extra Nuclear DNA of Eukaryotes - Eukaryotic Genomes
DNA molecules of MITOCHONDRIA and CHLOROPLASTS; resemble chromosome of prokaryotes; code for ~5% of RNA & proteins - Some fungi & protozoa carry plasmids - these are in the NUCLEUS
Mutants
Descendants of a cell that does not repair a mutation
Nucleotide Analogs - Chemical Mutagens - Mutations of Genes
Disrupt DNA & RNA replication
Horizontal Gene Transfer - Prokaryotes
Donor cell contributes part of genome to recepient cell 3 Types: 1. Transformation 2. Transduction 3. Bacterial Conjugation
Genetic Recombination & Transfer
Exchange of nucleotide sequences often occurs between homologous sequences
rRNA
Makes up the Ribosome
Participants in Translation
Messenger RNA: this is translated Transfer RNA: this is used to decode, carries AA Ribosomes: this does the translating
Nuclear Chromosomes - Eukaryotic Genomes
More than 1 chromosome/cell (humans=46); Chromosomes are LINEAR & sequestered within nucleus; Eukaryotic cells = Diploid (2 chromosome copies)
RNA Primers
Necessary for DNA replication
Prokaryotic Operons - Genetic Expression
Operon consists of a promoter and a series of genes - controlled by a regulator element called an operator - operators control operons
Vertical Gene Transfer
Organisms replicate their genomes and provide copies to descendants
Phenotype
Physical features and functional traits of the organism
Events in Transcription
Prokaryotes - Occurs in Nucleoid Eukaryotes - Occurs in Nucleus 1. Initiation - START 2. Elongation - RUN 3. Termination - STOP
Riboswitch - Control of Translation
RNA molecule binds a molecule, which causes a shape change to regulate translation or transcription
Short Interference RNA (siRNA) - Control of Translation
RNA molecule complementary to a portion of mRNA, tRNA, or DNA that binds and renders the target inactive
Types of Plasmids
Resistance Factors Bacteriocin Factors Virulence Plasmids
Nucleotide-altering Chemicals - Chemical Mutagens - Mutations of Genes
Result in base-pair substitutions and missense mutations
Frameshift Mutagens - Chemical Mutations of Genes
Result in nonsense mutations
Genotype
Set of genes in genome
Prokaryotic Transcription - Elongation
Sigma is lost and transcription occurs making a complementary RNA strand
Genetics
Study of inheritance and inheritable traits as expressed in an organism's genetic material
Anti-Parallel Synthesis
Synthesis for both strands is in the same direction
Genome
The entire complement of an organism; including its genes and nucleotide sequence
Nonionizing Radiation - Mutagens - Mutations of Genes
UV radiation
microRNA - Control of Translation
bind complementary mRNA & inhibit its translation
mRNA
messenger RNA -- carry code to make proteins, translated
Regulator RNA
shRNA, miRNA, piwiRNA, snRNA, snoRNA
tRNA
transfer RNA; charged with Amino Acids, used for translation