-physical features and functional traits of an organism
-reflection of proteins it possesses at any point in time
-proteins are grouped into functional categories
-particular set of genes an organism possesses
-organism’s genotype resides in its genome
Gene
unit of genetic information
segment of DNA that possesses information specifying the order (= sequence) of amino acids in a polypeptide molecule
sequence of nucleotides in a stable RNA molecule (tRNA or rRNA)
not all genes specify polypeptides
some genes specify rRNA or tRNA as their final gene product
Genome
total physical DNA that specifies an organism’s characteristics → all of its genetic materia
composed of DNA making up an organism’s gene and DNA between its genes
genomics — study of genomes
bacterial genomes constitute its chromosome and plasmids
Proteome
all of the different proteins that can be synthesized by an organism
proteomics — study and analysis of proteomes
Chromosome
eukaryotes
chromosomal DNA is linear
organellar DNA is circular
prokaryotes
genome components are circular DNA
DNA replication is initiated by proteins which bind at a specific nucleotide sequence — origin of replication oric
DNA synthesis proceeds in both directions (bidirectionally) around chromosome to a termination region opposite oriC on the chromosome
all prokaryotic genomes must have the same general classes of chromosomal genes
eg. genes encoding structural proteins, enzymes, transport proteins, DNA replication proteins, proteins for transcription and translation, regulatory DNA binding proteins, genes encoding tRNAs and rRNAs
different organisms have different mixes of genes within these classes (genotype) that endows them with particular characteristics and abilities (phenotype) linked to microenvironments they have adapted to
-bacterias contain genes outside their chromosome
-small circular pieces of DNA
-most plasmids not essential for viability of host under all environmental conditions but can augment a prokaryote’s ability to survive in certain circumstances
-often grouped according to genes they carry
characteristics they give host organism in which they exist
-prokaryotic cells can host single or multiple copies of different plasmids at the same time
-acquired via horizontal gene transfer
can be acquired by a cell without it needing to divide so genotype of an existing cell can change without it needing to reproduce
-theta bidirectional
many plasmids are replicated like the chromosome
beginning at origin of replication with specific nucleotide sequence (oriV) followed by bidirectional complementary DNA synthesis around dsDNA circle
Resistance Plasmids
carry genes specifying proteins that confer resistance to antibiotics and other toxic chemicals
Nutrition Plasmids
carry genes which specify enzymes that allow organisms to use certain molecules as nutrients
molecules sometimes unusual chemicals (petroleum, pesticides) in the environment that are not essential for survival
Infection Plasmids
carry genes which specify proteins that are not required for establishment of infections in animals and plants
Symbiosis Plasmids
carry genes which specify proteins that are required for establishment of symbiotic relationship with plants and animals
-most prokaryotic genes are protein encoding
specifies a polypeptide rather than a stable RNA
-typical bacteria gene consists of
promoter region
RNA polymerase binds during transcription
operator region
regulatory protein can bind
depending on type of regulation → promotes or inhibits transcription
coding region
contains the template for mRNA
terminator
RNA polymerase falls off the DNA to end transcription
-prokaryotic cells have evolved to have operons
-gene clusters
-multiple genes under the control of a single promoter
-common to have genes clustered together in the genome if they specific proteins that are involved in the same cellular processes
-genes of operons are transcribed together as a single mRNA initiating at one promoter but 2 or more different proteins with different amino acid sequences are translated from the single mRNA
-transcription begins at a promoter, region of dsDNA about 50 bp in size
-promoters have specific nucleotide sequences that RNA polymerase recognizes to initiate transcription
RNA Polymerase
bacteria have a single RNA polymerase
binds nonspecifically to DNA and slides along it scanning for promoter sequence
once detected, polymerase stops → transcription initiated → sigma subunit dissociates
exists in 2 forms:
holoenzyme
core enzyme
4 subunits
2 alpha subunits
2 beta subunits
must associate with a 5th protein (sigma factor) to form holoenzyme
-sigma subunit confers the ability to recognize a promoter sequence
different sigma subunits have different sequence specificity for promoter recognition
allows bacteria to regulate the transcription of entire categories of genes by controlling whether particular sigma factors are made
-coding region acts as the template for mRNA
-after RNA polymerase binds to promoter → RNA polymerase separates DNA strands to gain access to nucleotides of template strand
-enzyme moves along DNA strand in 3’ to 5’ direction
synthesizes mRNA in 5’ to 3’ direction
uses nucleotide sequence of template strand as guide for choosing complementary nucleotides
-first nucleotide copied into mRNA (+1 site) occurs a few nucleotides downstream of promoter
-intrinsic termination
RNA polymerase falls off template strand when it pauses over a stretch of weakly H-bonded A-U nucleotide pairs
RNA polymerase pausing associated with formation of stem-loop structure in mRNA that interacts with enzyme
-intrinsic transcription terminator — stem-loop structure
-nucleotide sequence of mRNA is used by ribosomes as a guide to link amino acids together in a particular sequence resulting in a unique polypeptide
-for translation to begin, ribosomes specifically bind to mRNA at a nucleotide sequence (ribosome binding site - rbs)
varies somewhat in sequence depending on gene but all are ~5 nucleotides in length
-after binding to mRNA at rbs
with participation of tRNA → ribosomes move along mRNA in the 5’ to 3’ direction joining amino acids together via peptide linkages in the sequence specified by nucleotide sequence in mRNA
-several ribosomes can sequentially bind to and simultaneously translate the same mRNA
-translation can start even before mRNA synthesis is finished
-during translation, nucleotides are read by ribosomes and tRNA in groups of three (codons)
codons specify the amino acids according to the genetic code
-polypeptides are synthesized from the N-terminus (first) to C-terminus (last) so 5’ end of mRNA coding region corresponds to N-terminus of polypeptide and 3’ end of mRNA coding region corresponds to the C-terminus
-when ribosome encounters stop codon (UAG, UAA, UGA) translation stops and polypeptide is complete
-proteins are expensive to make
-formation of each peptide linkage consumes 5 ATP and proteins have hundreds of amino acids
-translation of a single protein requires thousands of ATP molecules
-controlling when, where and how much of a particular gene product is made
Constitutive (Housekeeping) Gene Expression
some genes expressed at all times because product of gene is required for a basic cellular function required at all times
Developmentally Regulated Gene Expression
some genes must be expressed at different phases of a process or growth so that different proteins are made at different times
Cell-Specific Gene Expression
all cells that make up prokaryotic organism contain the same genetic information
some genes are expressed only in certain cell types
Environmentally Regulated Gene Expression
some genes are expressed only under certain environmental conditions
-frequency in which RNA polymerase initiates transcription
-strong promoters initiate transcription more frequently than weak promoters
strong promoters = RNA polymerase spends more time bound to DNA = higher binding affinity = greater likelihood transcription is initiated = gene transcribed and encoded polypeptide synthesized
-for a particular sigma subunit → all promoters have similar nucleotide sequences to an extent
different RNA polymerase binding characteristics
-recruits or inhibits RNA polymerase by actively binding to operator region of promoter
Lac Operon Example
lac operon has genes that express enzymes involved in lactose metabolism
cell only expresses proteins when lactose is the substrate
wasteful for cell to express proteins when there is no lactose to metabolize
lactose molecules must help regulate when lac operon is expressed or not
LacI gene is upstream of lac operon
encodes for transcriptional regulator
transcriptional repressor → binds to operator region that’s downstream of promoter (site where RNA polymerase and sigma factor binds)
Lactose absent
LacI bound to operator preventing expression of lac operon
Lactose present
LacI regulator binds to lactose → changes conformation → LacI can’t bind to operator → RNA polymerase able to transcript lac operon genes → express lac operon enzymes
CAP Site Example
upstream of lac promoter is CAP site
cAMP binds to CAP → changes conformation → able to bind to CAP site
CAP is an enhancer that recruits RNA polymerase
promotes expression of lac operon
amount of glucose in environment inversely related to amount of cAMP produced in cell
low glucose → cAMP and CAP binds to CAP site → RNA polymerase binds to promoter → high transcription
high glucose → no cAMP → CAP can’t bind to CAP site → basal levels of transcription
Regulatory | Operon Binding Site | Relationship to Environment | Overall Outcome |
LacI | Lac promoter | lactose binds LacI and inhibits repression effect | high lactose → no lac operon repression |
CAP | CAP site | glucose downregulates cAMP → cAMP binds CAP and increases repression effect | high glucose → lac operon repression |
-enhances transcription
-inhibits transcription
-mRNA is subject to enzymatic degradation by ribonucleic enzymes (RNAses) as soon as it’s made
-mRNA = unstable RNA
-different mRNAs have different half-lives in prokaryotic cells
-some have half lives of ~1 minute while others have a half-life ~1 hour
-longer mRNA lasts in cell → more polypeptide copies can be translated from it
-only a few copies of a polypeptide may be translated from a short-lived mRNA whereas 100s of copies of polypeptide may be translated from a long-lived mRNA
-most costly mean of regulating gene expression
-directly degrade proteins that cell doesn’t need
-costly because cell has already spent resources to express and produce protein
-proteins meant for degradation → tagged and sent to large protease complex to be recycled back into their peptide or amino acid forms
-organisms obtain genes from nonparental sources
-organism that donates DNA = donor
-organism that receives DNA = recipient
-transport of naked donor DNA from environment into recipient
-bacteria dies → lyse → release DNA and other cytoplasmic contents into environment
-DNA breaks into 100s of linear pieces/fragments
average fragment carried ~20-100 average genes
fragments can be taken up by other recipient cells
-bacteria have to be in competent state to transfer DNA from the environment
proteins for transport of dNA from environment are synthesized
-com proteins — proteins or transport of DNA
-transfer of DNA from donor to recipient mediated by virus (bacteriophage)
result in change in phenotype of recipient
-binding of phage particle to cell surface receptor initiates infection process
-phage DNA injected into host cell cytoplasm and remained of T4 phage particle remains on cell surface
-depending on conditions and type of phage → bacteriophage can undergo 2 lifecycles
lytic life cycle
phage forces host to express its own viral proteins allowing for virus particles to assemble inside the host
virus lyses and releases newly formed particles into environment
lysogenic life cycle
integrates its viral DNA into host chromosome and stays dormant until certain conditions trigger lytic life cycle
-when new phage particles are being packaged in host → there’s a chance that fragmented host (bacterial DNA) is packaged into phage head by mistake
transduction can occur because phage containing accidental bacterial DNA can go and infect another bacterial cell and transfer that DNA to a new host
-transducing phage — phage particle containing only host DNA
Bacteriophage
infectious particles of genetic material wrapped in protein coat
tailed dsDNA phage particles possess
protein capsids (heads)
coiled bodies
injects DNA into bacterial host
-transfer of plasmid from one prokaryote to another via cell-to-cell contact
-plasmids are genetic elements that can infect host cells
exist only inside prokaryotic cells
don’t have an extracellular state
-~25% of plasmids can directly transfer themselves from one organism to another via cell-to-cell contact and during conjugation
-plasmid-containing organism (donor) and organism receiving plasmid (recipient)
-conjugative plasmid possess DNA transfer or tra genes that encode transfer (Tra) proteins required to mediate the conjugative transfer of plasmid DNA from one cell to another
-initial contact between donor and recipient is made with conjugation pili
-proteins that compose conjugation pili are specified by some of the tra genes of conjugative plasmid in donor
-Type IV pili
-F-pili
-P-pili
-plasmids can be introduced into cell via conjugation or transformation
-plasmids are stable → independently replicating entities can work autonomously from chromosome
-degraded by nucleotides via nucleases specific for ssDNA normally present in bacterial cells
-sometimes ss donor DNA fragments escape degradation because they interact with RecA protein
potential for donor DNA to become integrated into recipient chromosome
-depends on whether donor DNA and recipient chromosomal DNA share homology
-2 regions of DNA exhibit high degree of nucleotide sequence similarity → homologous
-if 2 molecules of DNA share a region of homology of at least a few hundred nucleotides
2 molecules can be joined together at region of homology by homologous recombination
mediated by RecA protein
endonuclease (nickase) and ligase activities are involved in the process
allows DNA fragment to incorporate itself into chromosome and genes may be expressed to alter or affect host phenotype