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