Exam #2 Flashcards
Genome
genome: complete set of genetic information
chromosome plus plasmids
all cells: DNA
(viruses may have RNA)
functional unit is gene
encodes gene product, usually a protein
study of nucleotide sequence is genomics
bacterial genome
bacterial chromosome: circular molecule of DNA
- a self-replicating genetic element
extra-chromosomal genetic elements: plasmids
nonessential replicons
resistance to antimicrobial agents or production of virulence factors
Central dogma
The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
DNA
Incredible diversity of life determined by information within DNA
composed of four nucleotides:
adenine (A), thymine (T), cytosine (C) , guanine (G)
DNA can code for enormous amount of information
3 nucleotides encode specific amino acid
amino acids make up protein
sequence determines structure
DNA, RNA initially synthesized as ribonucleotides
purines: atoms added to ribose 5-phosphate to form ring
pyrimidines: ring made, then attached to ribose 5-phosphate
can be converted to other nucleobases of same type
Purines: Adenine Guanine double ring
Pyrimidine: Thymine Cytosine Uracil single ring
Base pairing
nucleotides joined between 5′PO4 and 3′OH with ester link
forms sugar-phosphate backbone
single DNA strand will have a 5′ and 3′ end
strands are complementary and antiparallel
held together by hydrogen bonds between nucleobases
base-pairing:
cytosine (C) to guanine (G) (three hydrogen bonds)
adenine (A) to thymine (T) (two hydrogen bonds)
separating strands called melting or denaturing
characteristics of RNA
RNA (ribonucleic acid)
ribose instead of deoxyribose
uracil in place of thymine
usually shorter single strand
synthesized from DNA template strand
RNA molecule is transcript
base-pairing rules apply except uracil pairs with adenine
transcript quickly separates from DNA
characteristics of RNA
RNA (ribonucleic acid)
three types required for gene expression
messenger RNA (mRNA)
ribosomal RNA (rRNA)
transfer RNA (tRNA)
DNA replication
DNA replication usually bidirectional
creates two replication forks
ultimately meet at terminating site when process complete
replication is semiconservative
In the two new molecules generated, each has one new strand and one original strand
replication begins at origin of replication
proteins recognize and bind to site
melt double-stranded DNA
oriC region characteristics
Replication is initiated through cooperative binding of the initiator protein, DnaA, to multiple DnaA-recognition sites within the oriC region.
SeqA strictly prevents the initiation of new rounds of replication via a mechanism called “sequestration.” SeqA inhibits replication initiation by blocking DnaA from binding.
Fis negatively influences replication initiation by regulating the occupation of DnaA.
IHF binding leads to bending of the DNA.
This triggers separation of the DNA strands at the AT-rich DNA unwinding element (DUE), providing an entry site for helicase and later on the other enzymes (e.g., primase and DNA Pol III) that are responsible for DNA synthesis.
In circular DNA, bidirectional replication from an origin leads to the formation of replication intermediates resembling the Greek letter theta.
Primase
primases synthesize short stretches of complementary RNA called primers
At ORI site, two leading strands primed, one in each direction
Primers are required for DNA synthesis because no known DNA polymerase is able to initiate polynucleotide synthesis. DNA polymerases are specialized for elongating polynucleotide chains from their available 3′-hydroxyl termini. In contrast, RNA polymerases can elongate and initiate polynucleotides.
Primer: initiation of DNA synthesis
process of DNA replication
DNA polymerases synthesize in 5′ to 3′ direction
hydrolysis of high-energy phosphate bond powers
DNA polymerase can only add nucleotides, not initiate
require primers at origin of replication
helicases “unzip” DNA strands
reveals template sequences
leading strand synthesized continuously
lagging strand synthesized discontinuously
DNA polymerases can only add nucleotides to 3′ end
production of Okazaki fragments
different DNA polymerase replaces primers
DNA ligase forms covalent bond between adjacent nucleotides
bacterial chromosome
Origin and terminus of replication divide genome into oppositely replicated halves
1 – replicated clockwise
has presented strand of E. coli as
leading strand
2 – complementary strand is leading one.
Transcription
RNA polymerase synthesizes single-stranded RNA
uses DNA template
synthesizes in 5′ to 3′ direction
can initiate without primer
binds to promoter
found upstream of genes
stops at terminator
transcription ends
transcription
RNA polymerase uses DNA template to synthesize single-stranded RNA transcript in 5’ to 3’ direction
transcription
RNA sequence is complementary, antiparallel to DNA template strand
DNA template is minus (–) strand
complement is plus (+) strand
RNA has same sequence as (+) DNA strand except uracil instead of thymine
mRNA transcripts are MONOCISTRONIC (code for one gene)
OR
POLYCISTRONIC (code for multiple genes)…
Sigma (σ) factor recognizes promoter
subunit loosely attached to RNA polymerase
various types of sigma factors recognize different promoters
synthesis controls transcription of sets of genes
eukaryotic cells, archaea use transcription factors
Initiation of transcription begins with promoter binding by RNA polymerase holoenzyme.
holoenzyme = RNA polymerase core + sigma factor
Promoters
promoter orients the direction of transcription in one of two directions.
By doing so, it also determines which strand is the template for the transcript.
found upstream of genes
once RNA polymerase has moved past, another RNA polymerase can bind
allows rapid and repeated transcription of single gene
Operon
remember: bacteria may make polycistronic (polygenic) mRNAs
An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter.
Why is knowing the orientation of a promoter critical when determining the amino acid sequence of an encoded protein?
The promoter orients the RNA polymerase in one of two directions.
By doing so, it also determines which strand is the template for the transcript.
Translation
genetic code: three nucleotides = codon
redundancy: code is degenerate
three reading frames possible
depends on start of coding region
correct reading frame is critical
incorrect will yield different, likely nonfunctional protein
translation in prokaryotes begins before transcription is complete
Ribosomes and translation
ribosomes serve as translation “machines”
prokaryotic comprised of 30S and 50S subunits
made from protein and ribosomal RNA (rRNA)
locate punctuation sequences on mRNA molecule
begins at start site, moves along in 5′ to 3′
maintain correct reading frame
aligns and forms peptide bond between amino acids
tRNA
transfer RNA (tRNAs) deliver correct amino acid
-has specific anticodon sequence
-base-pairs with correct codon
-carries appropriate amino acid
after delivering, tRNA can be recycled
enzyme in cytoplasm recognizes tRNA and attaches appropriate amino acid
translation initiation
part of ribosome binds to mRNA sequence
termed *ribosome-binding site
first AUG after that site serves as start codon
complete ribosome assembles at start codon
initiating tRNA brings altered form of methionine
occupies P-site
(peptidyl-site)
Ribosome has two sites to which tRNAs can bind
P-site occupied by tRNA carrying methionine
another tRNA recognizes codon in empty A-site
occupies A-site, brings correct amino acid
A-site and P-site now occupied by correct tRNAs
enzyme creates peptide bond between their amino acids
amino acid from tRNA in P-site added to amino acid carried by tRNA in A-site
Elongation (translation)
elongation of polypeptide chain
ribosome advances along mRNA in 5′ to 3′ direction
initiating tRNA exits through E-site
remaining tRNA carrying both amino acids occupies P-site
A-site transiently empty
a tRNA that recognizes codon in A-site quickly attaches
peptide bond formed between amino acids
ribosome advances one codon on mRNA
tRNA exits E-site, new tRNA occupies A-site
process repeats
once ribosome clears initiating sequences, another ribosome can bind: polyribosome, or polysome
Termination (translation)
termination
elongation continues until ribosome reaches stop codon
not recognized by tRNA
enzymes free polypeptide
break covalent bond joining to tRNA
freed ribosome falls off mRNA
disassociates into component subunits (30S and 50S)
subunits can be reused to initiate translation at other sites
Amino acid synthesis much slower than DNA synthesis
signal transduction
The regulation of gene expression is influenced by external and internal molecular cues and/or signals.
Sensing and responding to environmental fluctuations
Single-celled organisms with short doubling times must respond extremely rapidly to their environment.
Bacteria are exposed to changing conditions and must be able to adapt to stresses such as nutrient limitation, temperature shifts, varying osmolarity, and transition from exponential growth to stationary phase
Adaptation involves changes in gene expression
signal transduction
transmits information from outside cell to inside
allows cells to monitor and react
Regulation of protein expression in bacteria mostly occurs at the level of transcription of genes, carried out by RNA polymerase (RNAP) by binding to specific regions on the chromosome (promoters).
Quorum sensing
microorganisms constantly face changing environment
must adapt quickly to survive
quorum sensing
some organisms can “sense” density of their population
allows cells to activate genes useful with critical mass
for example, biofilm formation, pathogens′ infective process
quorum sensing is a method of cell-to-cell communication
affects gene expression
and physiological behavior of microbial communities.
Production of signal molecules continuous for each cell, BUT…responses only initiated when signal reaches threshold concentration.
quorum of bacteria required to produce signal concentrations above threshold; as population increases, concentration of signal increases because more signal producers present
some can detect, interfere with signaling molecules of other species
can “eavesdrop” and obstruct “conversations”
examples:
bioluminesence
biofilm formation
pathogens′ infective process
quorum sensing - Pseudomonas aeruginosa
Gram-negative
capable of surviving in wide range of environments.
opportunistic pathogen
commonly associated with nosocomial infections
burn wound infections
leading cause of death in severe respiratory infections (cystic fibrosis)
quorum sensing has key role in pathogenesis of P. aeruginosa
regulates production of extracellular virulence factors
regulates expression of antibiotic efflux pumps
promotes biofilm maturation
Infections with P. aeruginosa difficult to eradicate, due to antibiotic resistance and growth in biofilms.
biofilm formation
switch from single-cell, planktonic lifestyle to multicellular, sessile biofilm involves changes in gene expression to produce:
adhesins
extracellular polysaccharide-containing matrix
flagella formation
quorum sensing in Gram positive cells
Quorum sensing in Gram positive cells – small peptides (autoinducing peptides)
Peptide signals are not diffusible across the membrane
two-component regulatory systems
membrane-spanning sensor
modifies internal region in response to
specific environmental variations
phosphorylates amino acid
histidine kinase sensor protein
response regulator
phosphate group transferred from sensor
regulator turns genes on or off in response
and a transcriptional regulator known as response regulator
Natural selection
natural selection can play role in gene expression
expression of some genes changes randomly in cells
enhances survival of at least part of population
Antigenic variation
antigenic variation - alteration of characteristics of surface proteins - allows pathogens to stay ahead of host defenses
Neisseria gonorrhoeae - many genes for pilin (protein subunit of pili)
only expresses gene in expression locus
randomly moves genes in and out of expression locus
immune system responds to dominant pilin type
bacteria that have “switched” type survive
Phase variation
phase variation involves switching genes on and off
allows E. coli to attach via pili, detach by turning off
regulation of gene expression
-except for “house-keeping” genes, most genes expressed or repressed depending on specific conditions under which cells grow.
-competition for scarce resources (nutrients) makes bacterial cells efficient
Regulating gene expression is one way to save energy.
-gene activity controlled by promoter and regulatory elements that determine whether RNAP will transcribe gene
bacterial gene regulation
Genes can be routinely expressed or regulated
a set of regulated genes transcribed as single mRNA along with its control sequences is termed operon
-lac operon for lactose metabolism
separate operons controlled by single regulatory mechanism constitute regulon
global control is simultaneous regulation of numerous genes
Type of regulation
enzymes can be grouped by type of regulation
constitutive enzymes synthesized constantly
typically indispensable roles in central metabolism (enzymes of glycolysis)
inducible enzymes not routinely produced
Synthesize only when needed (β-galactosidase turned on only when lactose present)
avoid waste of resources
repressible enzymes
produced routinely
turned off when not required
(anabolic pathways such
as amino acid synthesis)
What is the difference between inducible gene expression and repressible gene expression?
Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression.
Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.
mechanisms to control transcription
must be readily reversible, allow cells to control relative number of transcripts produced
two most common are alternative sigma factors and DNA-binding proteins
Sigma factors
alternative sigma factors
standard sigma factor (σ70 - sigma factor with molecular weight of 70 - “housekeeping” sigma factor or primary sigma factor, transcribes most genes in growing cells. Every cell has a “housekeeping” sigma factor that keeps essential genes and pathways operating)
is loose component of RNA polymerase that recognizes specific promoters for genes expressed during routine growth conditions
Alternate sigma factors recognize promoters of different architectures – different regulons of different genes; with alternative sigma factors RNAP redirected to new sets of genes
Sigma 70 - housekeeping sigma factor - association with RNAP favored because high intracellular level and higher affinity to core RNAP
Most housekeeping genes expressed during exponential growth transcribed by holoenzyme containing σ70 and RNA polymerase
Alternative σ factors provide a line of response to fluctuating changes in their environment such as heat shock, variation in pH, and osmolarity, nutrient deprivation by effectively reprograming the transcription of sets of specific genes
-sporulation in Bacillus subtilis controlled by multiple different alternative sigma factors
DNA binding proteins: repressors
DNA-binding proteins can act as repressors or activators
repressor blocks transcription (negative regulation)
binds to operator, stops RNA polymerase
repressors are allosteric: have binding site that alters ability to bind to DNA
two general mechanisms
induction: repressor binds, blocks transcription
inducer binds to repressor, repressor unable to bind
Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression.
repression: repressor unable to bind to DNA
corepressor attaches to repressor, complex now binds to DNA and blocks transcription
Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.
DNA binding proteins: activators
activator facilitates transcription (positive regulation)
ineffective promoter preceded by activator-binding site
binding of activator enhances ability of RNA to initiate transcription at promoter
inducer binding to activator allows binding to DNA///
Transcription control
DNA-binding proteins can act as repressors or activators
repressor blocks transcription (negative regulation)
binds to operator, stops RNA polymerase
repressors are allosteric: have binding site that alters ability to bind to DNA
two general mechanisms
induction: repressor binds, blocks transcription
inducer binds to repressor, repressor unable to bind
repression: repressor unable to bind to DNA
corepressor attaches to repressor, complex now binds to DNA and blocks transcription
lac operon –
inducible gene expression
lactose and the lac operon
no lactose: repressor prevents transcription
lactose present: some converted to inducer allolactose
binds to repressor
repressor releases
operator
RNA polymerase
transcribes operon
only occurs when
glucose unavailable
mechanisms to control transcription – lac repressor
No lactose: lac operon proteins not made, because not needed.
If lactose, inducer binds to lac repressor, allows transcription of lac operon genes.
When lactose depleted, lac repressor loses inducer, and blocks production of proteins, since they are no longer needed.
Glucose and the lac operon
glucose and the lac operon
carbon catabolite repression (CCR) prevents expression of lac operon in presence of glucose
prioritize carbon/energy sources; yields diauxic growth
glucose transport system senses glucose
catabolite activator protein (CAP) required for transcription
functional only when bound by inducer cAMP
cAMP made when glucose low
Inducer exclusion: lactose transporter
blocked during glucose transport
cyclic adenine monophosphate (cAMP) involved in positive regulation of lac operon
transcription factor CAP (catabolite gene activator protein) forms complex with cAMP (inducer) and is activated to bind to DNA
cAMP level varies, depending on growth medium
cAMP low when glucose is carbon source:
glucose causes inhibition of adenylate cyclase, the enzyme that produces cAMP
Structural basis for cAMP-mediated allosteric control of the catabolite activator protein (CAP)
low glucose concentration
cAMP accumulates
binds allosteric site on CAP
CAP assumes active shape
binds upstream of lac promoter
makes it easier for RNA polymerase to bind promoter and start transcription of lac operon
increases rate of lac operon transcription
high glucose concentration
cAMP concentration decreases
CAP disengages from lac operon
high glucose concentration
cAMP concentration decreases
CAP disengaged from lac operon
low glucose concentration
cAMP accumulates
binds allosteric site on CAP
CAP assumes active shape
binds site upstream of lac promoter
makes it easier for RNA polymerase to bind adjacent promoter and start transcription of the lac operon
increases rate of lac operon transcription.
lac operon regulation by CAP (activator) and lac repressor
No lactose, no glucose
No glucose, lactose present
Glucose present, no lactose
Lactose present, glucose present
(Know the diagram)
Beta galactosidase
Beta galactosidase hydrolyzes lactose to produce glucose and galactose
(lac Z)
Lactose is both inducer for beta-galactosidase (lac Z) expression, and, when expressed, is its substrate
IPTG
experimental inducer - not hydrolyzed by beta-galactosidase
IPTG able to induce the operon, but it cannot be hydrolyzed by beta-galactosidase.
(IPTG can bind repressor to change its conformation so it will not bind at operator.)
induction of gene and performance of gene’s function can be separated.
genomics
analyzing a prokaryotic DNA sequence
(+) strand used to represent sequence of RNA transcript
ATG in (+) DNA indicates possible start codon
computers search for open reading frames (ORFs)
stretches of nucleotides generally longer than 300 bp
begin with start codon, end with stop codon
ORF potentially encodes protein
other characteristics (upstream sequence serving as ribosome-binding site) also suggest ORF encodes protein
can be compared with published sequences
presumed function can be assigned
Metagenomics
analysis of total microbial genomes in environment
can study all microorganisms and viruses in community
not limited to just those that grow in culture
can track changes in composition of microbiota of individual over time (healthy, diseased)
compare microbiota at different body sites
also between different individuals
study microbial life in oceans, soils
new understanding of extent of biodiversity
may lead to discoveries of useful compounds (for example, antibiotics)
Tremendous amount of data presents challenges
