Unit Questions

How do you define the genome of a prokaryote?

single circular dna molecule in nucleoid region

can include plasmids

In terms of the genome, how do archaea differ from bacteria, how are they the same?

both

• prokaryotes and no nucleus

• one circular chromosome

• dna in nucleoid region

• can have plasmids

differ

• gene structure and machinery: archaea gene and expression similar to eukaryotes

• dna packaging: archaea use histone like proteins to organize dna similar to eukaryotes

• rna polymerase: archaea have complex rna polymerase similar to eukaryotes; bacteria are more similar

How are genes organized in bacteria? Operon

group of genes controlled together

transcribed into single mrna

• structural genes - code for proteins and carry out cell functions

• promoter (start of transcription) - binding site for rna polymerase and located upstream

• operator (controls access or transcripton) - binding site for repressor protein

one switch controls multiple genes

How are genes organized in bacteria? Structural and control genes

structural

• code for proteins (enzymes) that do work in cell

• transcribed into mrna then translated into proteins

• worker

Control (regulatory)

• produce proteins (repressors/activators) that turn genes off/on

• regulate gene activity

• manager

How are genes organized in bacteria? Monocistronic and polycistronic mrna

monocistronic mrna

• one gene → one protein

• common in eukaryotes

Polycystronic mrna

• multiple genes → multiple proteins

• common in bacteria and operons

How are genes organized in bacteria? Regulon

group of genes or operons controlled by same regulatory protein

multiple operons controlled by one regulator

What are plasmids and what are some of their features?

circular double stranded dna molecules found in bacteria

own origin of replication (ori)

replicate independently

not essential for bacterial survival but beneficial

• r plasmids - antibiotic resistance genes

• virulence factors - toxin production genes

• metabolic genes - break down substances

moves via conjugation (cell to cell) to spread traits

How are plasmids maintained in a cell?

ori

• allows plasmids to copy themselves independently of bacterial chromosome

• ensures plasmid dna is available for daughter cells

regulate copies

• prevent over replication and maintains stability

partitioning

• par genes to ensure plasmids distributed to daughter cells during cell division

post par killing/ toxin antitoxin systems

• plasmids have toxin and antitoxin gene

• if plasmid is lost, antitoxin disappears and toxin damages or kills cell

selective advantage

• antibiotic resistance - plasmid cells survive antibiotics

How do low-copy-number plasmids and high-copy-number plasmids affect the cell and plasmid stability/expression?

low copy

• few copies (1-10)

• tight control systems and partitioning genes

• lower metabolic burden

• more stable

• low gene expression

high copy

• many copies (50-200+)

• rely on replication control

• higher protein expression

• higher metabolic burden

• slows growth of cell and adds stress

• unstable

What is rolling circle replication?

dna replication used by some plasmids bacteriophages and viruses to rapidly produce copies of circular dna

1. single strand nick - enzyme cuts strand of circular dna

2. 3’ is starting point - dna polymerase adds nucleotides to free 3’ OH end

3. strand displacement - new dna synthesized and old strand is unrolled

4. synthesis - circular template rolls, creating long single stranded dna tail

5. second strand synthesis - displaced single strand converted to double stranded dna and created multiple copies of original circular dna

What is transcription and what is the end result?

Copying genetic info from dna to rna

• carried out by rna polymerase and uses dna strand as template

rna polymerase binds to promoter and reads dna, building rna

What is translation and what is the end result?

making protein from mrna template

• occurs at ribosome

• translates mrna genetic code into amino acid sequence

1. mrna binds to ribosome

2. ribosome reads mrna in codons (3 bases)

3. trna bring matching acids

4. amino acids link to make polypeptide chain

5. folds into protein

What are -35 & -10 sequences, i.e., what is their significance?

dna regions in promoter of genes that help initiate transcription

• nucleotides upstream of transcription start site

rna polymerase

• recognized by sigma factor

• bind to promoter

-35 : recognition site

-10 : opening site (dna unwinding)

What component of RNA polymerase interacts with 010 and -35 sequences?

sigma factor

• recognizes and binds to promoter regions

helps rna polymerase

• locate promoter

• bind to dna

• initiate transcription

What is meant by promoter strength?

how effectively a promoter initiates transcription

• strong - rna polymerase binds frequently → high transcription → more mrna

• weak - rna polymerase binds less → lowtranscription → less mrna

How is basal level gene expression increased to high level expression?

basal - low transcription

positive regulation/activation increases expression

activator protein binds near promoter so rna polymerase binds more easily

What is the function of the Shine-Delgarno sequence? Where is it found?

ribosome binding site on mrna

• aligns ribosome with start codon

What is meant by vertical gene transmission and horizontal gene transmission? What are examples of how each of these processes occurs in prokaryotic cells?

vertical

• normal cell division - low genetic variation

• transfer genetic info from parent cell to daughter cell

• binary fission - dna replicated and split into identical cells

horizontal

• transfer of genetic material between cells not parent and offspring - high genetic variation

• can occur between diff bacteria or species

• conjugation - direct transfer via sex pilus (plasmids)

• transformation - uptake of free dna from environment (comes from dead/lysed cells)

• transduction - bacteriophages (infectious viruses)

What is the difference between core and flexible gene pools?

core

• genes in all strains of species located on main chromosome

• encode housekeeping functions for survival and reproduction

• stable pool and changes slowly (vertical)

flexible

• genes present in some strains found in mobile elements

• specialized functions for special niches/stress

• dynamic and updated through horizontal

What are genomic islands?

large segments of dna in genome acquired from other organism via HGT

provide upgrades for bacteria to adapt to environments

Describe the horizontal gene transfer mechanisms and components involved: Transformation; artificial v. natural transformation; Gram-negative cell vs. Gram-positive cell

transformation

• bacterium in competence state

• tightly regulated and triggered by stress or high cell density (quorum sensing)

competence factors

• signaling molecules that trigger expression of transformation genes

transformasome

• protein complex in cell membrane capturing extracellular dna and facilitates entry

translocasomes

• machinery that pulls dna strand into cytoplasm

gram positive

• uses transformasome

• non selective

gram negative

• move dna across 2 membranes (type iv pilus)

• highly selective

natural

• cell’s own encoded machinery (transformasome)

• spontaneously in wild

• Genetic diversity, DNA repair, or DNA as a nutrient source

artificial

• damage membrane to create temporary pores

• chemical - treated with CaCl2 followed by heat shock, neutralizing negative charge and creating pressure gradient

• electroporation - hit with high voltage electric pulse, destabilizing membrane and creating pores for dna entry

Describe the horizontal gene transfer mechanisms and components involved: Conjugation (include role/function of: sex pilus, fertility factor, Hfr formation, F prime factor)

f factor

• specialized dna containing genes for conjugation

• f+ are donors that have f factor

• f- are recipients lacking f factor

• codes protein needed to build conjugation bridge and enzymes to initiate dna transfer

sex pilus

• protein produced by donor cell

• pilus attaches to receptor on recipient cell and pulls cells together forming conjugation bridge

Describe the horizontal gene transfer mechanisms and components involved: Transduction. What other process of horizontal gene transfer does specialized transduction resemble and why?

transduction

• virus that infects bacteria carries dna from one cell to another

generalized

• lytic cycle - accidental packaging

• when phage is chopping host dna for its own, it puts bacterial dna instead of viral dna

• random gene

specialized

• lysogenic cycle where virus hides inside dna and waits

• viral dna integrates on bacterial chromosome and removes itself imprecisely, taking bacterial genes with it

• specific genes near viral site

Describe the horizontal gene transfer mechanisms and components involved: Transposition: what is this and how does this operate? What is conjugative transposition? Compare a simple transposon to a complex one. What are insertion sequences?

moving dna within cell - chromosome → diff spot

jumping genes because dna sequences jump around

insertion sequences

• carries bare minimum tools to move

• transposase genes - scissor enzyme cuts dna and puts it in new spot

  • inverted repeats - identical sequences at both ends as markers for transposase

simple transposons

• carry cargo genes between inverted repeats

complex transposons

• 2 separate insertion sequences to move everything in the middle

replicative

• copy paste - transposon replicated, one stays, one moves

non replicative

• cut past - transposon cut and moved

conjugative

• can jump out of chromosome of one cell, use conjugation, and jump into new chromosome in new cell

Describe the horizontal gene transfer mechanisms and components involved: What is a partial diploid cell?

carries normal set of genes plus second copy of specific genes

f’ conjugation

• f factor takes bacterial genes when exiting chromosome and new cell has original genes on chromosome plus second set of genes on new f’ plasmid (plasmid/sex pilus)

specialized transduction

• virus carries host genes to new cell and adds on to existing copies (virus)

What is the role of regulatory proteins in a bacterial cell?

control when and how genes are expressed

bind to dna regions (promoters/operators)

• activate expression → rna polym start transcription

• repress expression → block rna polymerase

Describe the levels at which control of gene expression can occur in a bacterium

transcription control

• controls if mrna is made from dna

• regulatory proteins turn genes on/off

• involves operons

post transcription control

• mrna stability and availability

• rna processing and modification

translational control

• mrna translation to protein

post translational control

• protein activation/inactivation

• protein folding, modification, and degradation

What are constitutive genes?

genes continuously expressed

not regulated or switched off

code for essential proteins

• metabolism

• cell structure

• dna replication

Describe the components of the operon model of gene organization and regulatory elements.

promoter

• DNA sequence where RNA polymerase binds and starts transcription

operator

• controls transcription

• regulatory proteins/repressors bind here

structural genes

• code for proteins

regulatory gene

• codes for regulatory protein

repressor

• binds to operator, blocks rna polymerase, and turns genes off

activator

• helps polymerase bind to promoter, increasing transcription

inducer

• inactivates repressor

corepressor

• activates repressor

What are inducible and repressible operons?

inducible

• turned on when inducer is present

• repressor active by default

• inducer then binds to repressor

repressible

• turned off when corepressor present

• inactive by default

• corepressor binds to repressor and activates it, stopping transcription

What is a corepressor?

turns off gene expression by activating repressor protein

What is an inducer?

turns ON gene expression by inactivating a repressor protein

What is derepression?

process of turning gene expression back ON by removing repression

Describe what the lactose operon is, and how the lactose operon functions in both the presence and absence of lactose.

inducible operon controlling genes needed to break down lactose

lactose (allolactose) acts as inducer and binds to repressor, inactivating it

How does cAMP-CRP complex affect expression of the lac operon? How does the presence of glucose affect the expression of the lac genes?

cAMP - cyclic AMP

CRP - catabolite activator protein

cells make more cAMP when glucose is low to bind to crp

binds near lac promoter

helps rna polymerase bind

increases transcription of lac genes

What is diauxic growth?

pattern of bacterial growth where cells grow in two distinct phases when two different sugars are available

first growth

• bacteria prefer glucose and growth is rapid

lag phase

• glucose runs out and bacteria stop growing

• adjust gene expression (activate lac operon)

second growth

• bacteria use lactose and activate lac operon

• growth resumes but slower than glucose

catabolite repression

• glucose inhibits cAMP-CRP and turns off lactose genes

Describe what the tryptophan operon is, and how the tryptophan operon functions in both the presence and absence of tryptophan.

repressible operon in bacteria that controls the genes needed to synthesize the amino acid tryptophan

• promoter

• operator

• structural genes

• regulatory genes

absence - cell makes own tryptophan

• repressor protein inactive and cannot bind to operator

• polymerase transcribes genes

• enzymes produced to synthesize tryptophan

presence - cell stops making tryptophan if available

• tryptophan is corepressor

• polymerase is blocked, blocking transcription, ceasing tryptophan

What is transcriptional attenuation? How does this work in the presence/absence of tryptophan?

gene regulation mechanism in bacteria that controls transcription

presence

• ribosomes move quickly and forms terminator hairpin

• polymerase stops transcription early

absence

• ribosomes stalls at codons in leader sequence

• anti terminator structure forms and polymerase continues transcription

What is phase variation? How does this work in the expression of Salmonella enterica flagellar protein?

expression of certain genes is reversibly switched ON and OFF in a random or controlled way

bacteria change surface structures to avoid immune detection and adapt to environments

What are small regulatory RNA (sRNA)? How do these function in regulation?

short RNA molecules that do not code for proteins but regulate gene expression at post transcriptional level

bind to target mrna molecules

• can repress or activate translation

What are anti-sense RNA’s? How do these function in regulating gene expression?

single stranded rna compllementary to mrna in bacteria

• can bind to mrna and affect expression

regulate genes by binding to mrna and blocking function

• block translation

• promote mrna degradation

What is the stringent response? How does this occur?

stress response when cell is nutrient limited

• helps survive harsh conditions by slowing growth

triggered by lack of amino acids, stress, or uncharged trna accumulate

• activation of relA

  • senses uncharged trnas at ribosome

  • synthesizes alarmone (ppGpp) and binds to polymerase

How do sigma factors figure into gene regulation?

proteins that help polymerase recognize and bind to promoters for transcription

control which genes are transcribed

• bind to core polymerase

• direct to promoter sequences