BIOL121: Lecture 7-10

4 levels gene expression is regulated:

• transcription

  • production of transcript

• post-transcription

  • stability or function of transcript

• translation

  • production of protein

• post-translation

  • stability or function of protein

() proteins are proteins are always required for growth

• housekeeping

() alter gene expression to help cell respond to conditions within or outside cell

• regulatory proteins/transcription factors

() is a group of related genes with a single promotor and a () is a group of genes/ () that are controlled by single regulatory protein

• operon

• regulon

• operon

Regulatory proteins come in 2 forms:

() to bind to regulatory sequences in DNA and prevent transcription of target genes

() to bind to regulatory sequences in DNA and stimulate transcription of target genes

Most () must first bind to small ligand

• repressors

• activators

• activators

2 types of repressors:

1. Bind operator DNA ()

   1. () binds to repressor causing it to release from () also called ()

2. Bind operator DNA ()

   1. () disappears, repressor releases from () and target gene can be expressed also called ()

1. by themselves

   1. inducer, operator, induction

2. only if corepressor is bound

   1. corepressor, operator, derepression

() are proteins that bind to promoters and can interact with () that’s stuck nearby to initiate ().

Some () don’t bind DNA activator sequences well unless () is present.

• activators, RNA polymerase, transcription

• inducer

The E. coli lac operon was discovered by () and ()

• Jacques Monod

• Francois Jacob

E. coli uses () for food but it can’t pass through plasma membrane () allows it entry and () is used to bring lactose inside cell.

• lactose

• lactose permease

• proton motive force

T/F: E. coli prefers glucose over lactose

• true

() encodes Beta-galactosidase

() encodes lactose permease

• lacZ

• lacY

T/F: Either lacZ or lacY is needed to digest lactose

• false

• both needed

T/F: Lactose digesting lacZYA operon of E. coli was the first gene regulatory system described

• true

How lactose is transported and metabolized

1. Cells use () made by () to transport lactose into cell

2. Cells use () made by () to either cleave lactose into () and () or alter lactose to produce ()

1. lactose permease, lacY

2. Beta-galactosidase, lacZ, galactose and glucose, allolactose

In the absence of lactose, () binds to () region and a DNA sequence bound by repressor () represses lac operon by preventing RNA polymerase from working well

• lacI

• operator

• lacO

In the presence of lactose, () made by () when at low concentrations rearranges lactose to make inducer () which binds to () reducing its affinity to the operator allowing operon to turn on

• beta-galactosidase

• lacZ

• allolactose

• lacI

In (), an operon enabling the break down of one nutrient is repressed by presence of more favorable nutrient

• catabolite repression

Maximum expression of lac operon requires presence of () and ()

() binds to promoter and interacts with RNA polymerase to increase rate of transcription ()

• cyclic AMP (cAMP), cAMP receptor protein (CRP)

• cAMP CRP, initiation

() is a sensor for glucose levels and glucose inhibits () production

• phosphotransferase system (PTS)

• cAMP

When glucose is present () are transferred to glucose making ().

IIA isn’t () so it inhibits cAMP production via ()

• phosphates, glucose-6-P

• phosphorylated, adenylate cyclase

When there is no glucose, PTS members are () meaning IIA doesn’t inhibit (), () is produced, and can activate lac operon

• phosphorylated

• adenylate cyclase

• cAMP

Glucose transport by PTS causes () by inhibiting LacY permease activity ()

• catabolite repression

• inducer exclusion

Genes encoding biosynthetic enzymes are regulated by repressors called () which bind the end product of the pathway, the ()

• inactive aporepressors

• corepressor

The aporepressor-corepressor complex, (), can bind to operator sequence upstream of target gene or operon.

Blocks () and so transcription is mostly off

• holorepressor

• RNA polymerase

The () is an operon that codes for many enzymes involved in tryptophan production

• tryptophan operon

When internal tryptophan levels exceed cellular needs, the excess tryptophan or () will bind to an inactive (), ().

The () then binds to an operator DNA sequence upstream of trp operon repressing the expression of structural genes by blocking ()

• corepressor, aporepressor, TrpR

• holorepressor, RNA polymerase

() is a mechanism to terminate transcription after it’s already started and is important in pathways where end products are ()

• attenuation

• low

If the concentration of tryptophan is high, the need for tryptophan biosynthesis genes is () and transcription and attenuation ().

If the concentration of tryptophan is low, the need for tryptophan biosynthesis genes is () and transcription and attenuation ().

If the concentration of tryptophan is extremely low, the need for tryptophan biosynthesis genes is () and transcription and attenuation ().

• low, transcription repressed, attenuation not mentioned

• moderate, transcription enabled, attenuation active

• high, transcription enabled, little attenuation

Leader sequences () control biosynthesis of tryptophan and () determine if RNA polymerase can transcribe into stuctural genes

• doesn’t

• does

Regulation of trp operon through attenuation is based on ().

There are 4 regions of ().

() stem loops are critical

Region 3:4 () forms when trp levels are () and RNA polymerase falls off RNA before it transcribes structural genes

Region 2:3 () forms when trp levels are ()

• RNA secondary structures

• mRNA

• 2

• attenuator loop, high

• anti-attenuator, low

Low tryptophan levels

1. Tryptophan levels dictate how many () present

2. Ribosome pauses at () because there’s () tryptophan

3. Allows region () loop to form

4. () loop can’t form so RNA polymerase transcribes trp genes

1. trp-tRNAs

2. leader codons, no

3. 2:3 anti-attenuator

4. attenuator

High tryptophan levels

1. With lots of tryptophan present, ribosome () trp codons then a stop codon

2. Ribosome stops and physically covers region ()

3. This allows () loop to form

4. () loop releases RNA polymerase before it can transcribe ()

1. translates

2. 1:2

3. 3:4 attenuator

4. attenuator, trpE

() converts arabinose into () an intermediate in a biosynthetic pathway

• arabinose operon

• xylulose-P

T/F: Major regulator AraC represses gene expression

• false

• Major regulator AraC can repress or activate gene expression depending on whether substrate is available

When arabinose is (), AraC shape is (). It represses expression of genes that break down arabinose.

When arabinose is (), AraC shape is (). It stimulates binding of RNA polymerase to transcribe genes.

• absent, rigid and elongated

• present, compact

When arabinose is absent, the N-terminal arm binds to ()

When arabinose is attached, the N-terminal arm binds to ().

• its own C-terminal DNA binding domain

• dimerization domain of other monomer

If no arabinose present, AraC acts as () and blocks ().

If arabinose is present, AraC acts as () and binds ().

• repressor, transcription

• activator, RNA polymerase

The () directs the expression of genes, operons, and regulons.

• alternative sigma factors

Sigma Factor Regulation: Heat-shock response

() encodes heat-response sigma factor ()

() controls expression of heat-shock response genes

Secondary structures at () end of rpoH mRNA blocks ribosome, reducing () translation

• rpoH, Sigma H

• Sigma H

• 5’

Sigma Factor Regulation: Heat-shock response

Under normal conditions, () is bound by chaperones and taken for () by DnaJ, DnaK, GrpE, also known as ()

Chaperones bind () proteins to try to fix denatured proteins

• Sigma H, degradation, anti-sigma factors

• heat-denatured

Sigma Factor Regulation: Heat-shock response

() controls levels of Sigma H

() melts RNA secondary structure and increases translation of ()

() causes proteins to misfold which draws chaperones away from () and allows it to promot transcription of ()

• heat

• heat, Sigma H

• heat, Sigma H, heat shock regulon

() are found within bacterial intergenic regions and regulate transcription or stability of mRNAs

• small regulatory RNAs

() nature of sRNA allow these molecules to bind mRNA which can either () mRNA or make it susceptible to ()

• antisense

• target

• degradation

Some () bind to () and block the ribosome which () translation or () translation

• sRNA

• ribosome-binding site

• turns off

• inhibits

Some () bind to () and free () to () translation or () translation

• sRNAs

• mRNA sequence

• Ribosome-binding site

• allow

• activate

Some () bind to an () and cause () to form. Cells don’t like () and this will turn translation () and promote ()

• sRNAs

• mRNA sequence

• dsRNA

• dsRNA

• off

• mRNA degradation

() refers to process where bacterial cells work together at high density and was discovered in () a bioluminescent bacterium that colonizes the light organ of Hawaiian squid

• quorum sensing

• vibrio fischeri

Induction of quorum sensing gene system requires accumulation of secreted small molecule called ()

• autoinducer

At a certain extracellular concentration, secreted autoinducer reenters cell and binds to () which in case of Vibrio fisheri is () and activates transcription of luciferase target genes that confer bioluminescence

• regulatory protein

• LuxR

What triggers burst in luminescence in V. fisheri?

() synthesizes () which diffuses out of cell

When a critical level is reached, () reenters cell and binds to ()

() complex activates transcription of luciferase genes that make luminescence

• LuxI, autoinducer

• autoinducer, LuxR regulatory protein

• LucR autoinducer

4 techniques to extract microbial DNA:

1. cells lysed with lysozyme to degrade cell wall, treated with detergents to dissolve membrane

2. proteins removed in high-salt solution

3. cleared lysate containing DNA passed through column containing silica resin that specifically binds DNA

4. extracted DNA examined with variety of analytic tools
   

PCR is used to () DNA sequences using () to target DNA sequence of interest and apply () and ()

• copy

• specific primers

• cyclical heat

• Taq, Thermus aquaticus DNA polymerase

() separates PCR products by size and lets you see them on agarose gel

• gel electrophoresis

() is an early sequencing method still in use today and uses a mixture of deoxy- and dideoxynucleotides to sequence DNA

• sanger sequencing

() sequencing builds chains of DNA and detects which base is added next

• illumina

() sequencing is part of () sequencing and sequences target PCR fragments

• amplicon

• illumina

() sequencing is part of () sequencing and sequences random fragments of DNA

• shotgun

• illumina

4 types of genetic manipulation of microbes:

1. mutagenesis

   1. random

   2. site-directed

2. targeted gene editing (CRISPR/Cas9)

3. restriction endonucleases

4. gene cloning

() is used to identify genes involved in microbial processes of interest.

A population of cells is exposes to a mutagen that alters genetic code at random locations within genome

• random mutagenesis

() is used when gene of interest is known.

The bacteria’s gene (allele) is replaced by plasmid containing different gene or allele.

• targeted (site-directed) mutagenesis

() is useful when you want to find genes involved in some processes

• isolating mutants

If a gene involved in acid resistance is knocked out then it () grow at low pH

• can not

() usually involves using antibiotic resistance genes in sequences that will interrupt the gene of interest creating ()

• generating mutants

• gene knockout

Isolating mutants

1. Randomly allow () to integrate

   1. screen mutants for desired phenotype

2. Specifically target a suspected gene of interest ()

   1. screen for correct recombination event

• transposon

• site directed mutagenesis

3 reasons why transposons should be easy to identify:

1. contains antibiotic resistance genes

   1. cells with transposons grow with antibiotics present

2. create large insertion mutations

   1. knock out gene function

3. easy to identify sequence

   1. mutated gene next to transposon

Create mutants by interrupting gene with ().

Identify mutated gene by using sequence DNA interrupted by (). Use known sequence of () to identify gene it interrupts.

• transposon x3

Targeted gene mutagenesis using () can knockout gene of interest with another gene

• homologous recombination

Introducing genes/DNA in bacteria on plasmid

Put gene of interest on () and transform () into () cell.

Plasmid will () once inside bacterial cell

• plasmid vector, vector into competent cell

• replicate itself

Regulation of gene can be determined by fusing () of gene of interest to () and it encodes an easily assayed protein like glowing fluorescent protein.

• promoter

• reporter gene

2 types of reporter fusions:

• operon fusion/transcriptional fusion

  • shows transcriptional control of gene

• gene fusion/translational fusion

  • shows transcriptional and translational control of gene

What is a virus?

• noncellular particle that must infect host to replicate

T/F: All viruses are obligate intracellular parasites which means they depend on host metabolism

• true

() is the virus particle and consists of nucleic acid and protein coat

• virion

T/F: Viruses sometimes have a protective protein coat called a capsid

• false

• always

Virus genomes only contain information for () and ()

• taking over host cell

• making viral proteins

The viral () packages the genome and delivers it into host cell.

The viral () is composed of repeated protein subunits

• capsid x2

Capsid + genome = ()

• nucleocapsid

Different viruses make 2 different capsid forms:

• symmetrical and asymmetrical

Viruses can be classified by 4 characteristics:

• shape

• structure

• genome composition

• replication mechanism

() have a structure that exhibits rotational symmetry and is a polyhedral with 20 identical triangular faces

• icosahedral viruses

The () is sometimes enclosed in an () formed from the host cell’s membrane

• capsid

• envelope

Between the envelope and capsid you’ll find () proteins

• tegument

() have a capsid that consists of a long tube of protein with genome coiled inside and vary in length depending on genome

• filamentous viruses

T/F: Icosahedral viruses include bacteriophages as well as animal and plant viruses

• false

• filamentous viruses?

In () capsid monomers form tube around genome

• filamentous viruses

() have complex multipart structures and include Tf bacteriophages that have () head and () neck

• asymmetrical viruses

• icosahedral

• helical

() virus has a genome that is surrounded by several layers including a core envelope studded with spike proteins and an outer membrane

• pox

Viral Life Cycle:

1. attach to host cell

2. get viral genome into host cell

3. replicate genome

4. make viral proteins

5. assemble capsids

6. release progeny viruses from host cell

Viral genomes

DNA/RNA can be:

Include genes encoding viral proteins:

• ss/ds, linear/circular

• capsid, envelope proteins, any polymerase not in hose

What Baltimore family is herpes and small pox?

1 dsDNA

What Baltimore family is parvovirus and geminiviruses?

2 ssDNA

What Baltimore family is rotavirus and reoviruses?

3 dsRNA

What Baltimore family is COVID, HepC, cold virus?

4 +ssRNA

What Baltimore family is flu, rabies, ebola?

5 -ssRNA

What Baltimore family is HIV?

6 RNA retrovirus, reverse transcriptase

What Baltimore family is HepB?

7 DNA pararetrovirus