MCB 150 Exam 4

cost of making the lacZ protein

DNA ~3,000 nt

transcribed to 3,000 nt mRNA (1 ATP per base = 3,000 ATP) translated to a 1,000 AA protein (# ATP per AA = 3,000 ATP)

- 6,000 ATP for 1 protein

rate of translation

20 AAs per second

1,000 AAs = 50 sec

6,000 ATP/50 sec = ~120 ATP used per second for one protein

glucose

preferred energy source in all organisms

gene regulation

turning on and off genes as they are needed in the cell to conserve energy

- adjusts metabolism to achieve maximum growth in a given environment

constitutive

genes that are needed at all times and are not regulated (except in M-phase when transcription ceases)

ex) CDKs

where can regulation occur

transcription initiation, transcript stability, translation initiation, rate of translation, activity of protein (phosphorylation), and polypeptide stability (protease)

where does regulation occur mainlY?

at transcription initiation (least amount of ATP is used, but trade off between energy savings and the speed at which the change takes effect)

operon

multiple protein-coding regions under the control of a single promoter

- transcribed as polycistronic mRNA (essentially the bacterial equivalent of alternative RNA splicing)

- different regions are independently translated to the different proteins

- each protein-coding region in the mRNA molecule has its own ribosome binding site and stop codon

ex) the lacZ, lacY, and lacA genes all make up th elac operon

lac operon in E. coli

produces beta-galactosidase, galactoside permease, and lac I

beta-galactosidase

- most abundant gene in the lac operon

- amount present varies on the accumulation of lactose in the cell

- breaks lactose down into galactose and glucose

at what level does the lac operon regulate gene expression?

at the transcription initiation stage

galactoside/lac permease

- lac Y

- allows lactose into the cell through the plasma membrane by creating holes in the membrane for it

what regulates the expression of the lac operon?

- glucose AND lactose

- glucose is used up first

- once there is no more glucose, a temporarily plateau in cell growth occurs while the cell reroutes its metabolic pathway

- lactose is then used to obtain glucose, leading to beta-galactosidase accumulating and cell growth resuming until no more lactose is present

components of the lac operon

Promoter, lac I, Operator, lacZ, lacY, and lacA

promoter

Region where RNA polymerase binds to initiate transcription.

lac I

not part of the operon, codes for the repressor that blocks RNA polymerase and prevents expression of the lac gene

operator

part of DNA where the lac repressor protein binds to to prevent RNA polymerase from continuing and transcribing the DNA

lacA gene

transacetylase protein, detoxifies toxic thiogalactosides that are accidentally let into the cell via lacY

lactose is also known as

allolactose

absence of lactose effects on the lac operon

lac repressor binds to the operator to prevent RNA polymerase from transcribing the operon

- negative regulation

presence of lactose on the lac operon

lactose bins to the repressor to let go and allow the lac operon to be transcribed by RNA polymerase

when does maximum transcription of the lac operon occur

when no glucose is present but lactose is present

- when glucose is present, lac is transcribed but inefficiently

positive regulation of lac operon

catabolite activator protein (CAP) or cAMP receptor protein (CRP) binds to the lac operon to induce transcription (binds to the promoter upstream of the operator)

as the concentration of glucose _____, the concentration of cAMP ______

increases; decreases

- glucose inhibits adenylyl cyclase

how does positive regulation of the lac operon occur

cAMP binds to the CRP and the complex binds to the promoter to enhance binding of RNA polymerase so transcription can occur

- lac operon is a weak promoter

what is a weak promoter

promoter with a sequence that is not similar to the consensus sequence, therefore RNA polymerase doesn't recognize it as frequently and transcription doesn't occur as much

process of lac operon activation

no glucose? inhibitor is removed by lactose binding which lifts negative regulation

cAMP and CRP complex binds to promoter sequence to apply positive regulation

what happens to the lac operon once all the lactose has been broken down?

beta-galactosidase shuts down transcription by breaking down the inducer (lactose), causing the lac repressor to rebind to the operon

can negative and positive regulation occur simultaneously?

yes; negative regulation beats positive regulation because it is located further down the gene by the start sequence, blocking RNA polymerase once it binds at the positive regulator

allolactose

product from lactose breakdown, binds to lac repressor and prevents it from binding to the operon by inducing a conformational change

levels of transcription of the lac operon

beta-galactosidase, permease, and transacetylase are transcribed in a 10:5:2 ratio

mRNA stability

- bacterial mRNAs have a half life of ~2 minutes to allow for a quick response to changes in the environment

- lac mRNA is degraded from the 3' end (partly explains the ratio of lacZ, Y, and A

translation initiation

- "strength" of the Shine-Dalgarno sequence (how close it is to the consensus) affects how much protein is made from the gene

- the lac operon SD for transacetylase is more hidden, explaining the ratio

- also affected by availability of SD sequence, which may be blocked by proteins or folded into secondary structure and unavailable

inducible operon

operon that is turned on when needed but is typically inactive

ex) lac operon

repressible operon

operon that is on by default and can be turned off when the target molecule is in abundance

ex) tryptophan

trp operon

- when tryptophan levels are low, the trp repressor is inactive, allowing trp operon to be transcribed

- when tryptophan levels are high, tryptophan (the corepressor) binds to the trp repressor, enabling it to bind to the operator site, which prevents transcription

- binding of corepressor causes a conformational change in the trp repressor allowing it to bind to the operator site and bock RNA polymerase from transcribing the operon

allosteric regulation with PFK

ADP = increase glycolysis rate

ATP bind = decrease glycolysis rate

regulation at the transcriptional level _____

saves the most energy

regulation at the post-translational level _____

takes effect faster

intermediate regulation

regulation between transcription and post-translational levels to make precisely the correct amount of functional protein under given conditions

core promoter in eukaryotes

sequence from -25 to +1 bases where general TFs bind to create the initiation complex, includes the TATA box

common locations for regulatory elements

proximal enhancers/silencers commonly between -100 and -50 bases from the start sequence

RNA polymerase II doesn't recognize _____ in eukaryotes

the promoter region: it needs transcription factors in order to bind

mediator function

connects gene-specific and general transcription factors to activate RNA polymerase

tissue/cell/gene specific transcription factors

TFs specialized in cells for certain genes

- can be close or far from the gene

general transcription factors

TFs present in all cells that are required for transcription to occur

- bind to core promoter and attract RNA polymerase to bind

- interact with mediator to connect with gene specific TFs

repressor proteins bind to ____ to ______

silencing sequence; stop transcription

activator proteins bind to _____ to ______

enhancer sequences; initiate transcription

effects of gene/tissue-specific factors (activators and repressors)

- affect ability of GTFs to bind core promoter sequence

- stimulate RNA polymerase II to proceed to elongation

- recruit chromatin-remodeling complexes

chromatin-remodeling complexes

- alter gene expression by changing chromatin or changing histones (tighter or looser hold on DNA)

GTFs allow ___ to bind but don't always allow ____

RNA polymerase; elongation to occur (requires additional proteins)

process of pre-initiation complex formation

1. activator binds to an enhancer in the promoter proximal region (GTFs can't bind until this does)

2. activator enhances the ability of the GTF TFIID to bind to the activator and the TATA box (immediately downstream of the enhancer, part of the core promoter region)

3. TFIID promotes the assembly of the preinitiation complex (more TFs and RNA polymerase and mediator) but elongation doesn't occur yet

elongation process

1. mediator binds to the preinitiaton complex, but transcriptional initiaton doesn't occur yet

2. activator binds to a distant enhancer and a coactivator binds to the activator. a bend in the DNA allows the activator-coactivator complex to interact with the mediator and cause RNA polymerase to proceed to the elongation stage

what does the mediator physically interact with

the coactivator-activator sequence on the distal enhancer and the preinitiation complex TFs

the tail of the mediator ____

activates RNA polymerase

what is required for RNA polymerase to elongate

mediator must be attached to pre-initiation complex and distal enhancer

what elements are required for transcription to occur?

all of them: GTFs, gene specific TFs, activators, coactivators, mediators, RNA polymerase, enhancers, core promoter sequences, promoter proximal sequences, etc

activators recruit _____

ATP-dependent chromatin remodeling complexes

functions of chromatin remodeling complexes

- change locations/spacing of nucleosomes

- evict histones from nucleosomes

- replace standard histones with histone variants

chromatin remodeling: change nucleosome positions

before remodeling: evenly spaced chromatin complexes

after remodeling: changes the relative positions of a few nucleosomes (some have more or less space between them) OR changes the spacing of nucleosomes over a long distance

chromatin remodeling: histone eviction

- chromatin-remodeling complex removes histones with chaperone proteins to allow for elongation

- histones are re-added to the DNA after the polymerase passes by and transcribes the DNA

chromatin remodeling: replacement with histone variants

- slightly different versions of H2A, H2B, etc that have looser or tighter binding effects to DNA can be interchanged to allow for the DNA to be held tighter or more loosely

H2A is replaced with another H2A, not with a completely different histone like H2B

histone modifying enzymes

enzymes that de/acetylate, de/methylate, or de/phosphorylate amino acids and cause different effects

ex) acetyltransferases, methylases, methyl transferases, demethylases, HAT (histone acetyltransferase)

covalent but reversible

acetylation tends to

loosen interactions between histones to increase transcription (enhance)

methylation tends to

tighten histone interactions to decrease transcription rates (silencing)

histone code

the transcription of DNA is partly regulated by the chemical modifications of histone proteins

- phosphorylating/demethylating/etc certain parts of a histone can determine which regions are transcribed and change what parts of the gene are expressed

NFRs

nucleosome free regions

- common in eukaryotes

- near promoters and the ends of genes/termination areas

- stretches of DNA with no chromatin

possible functions of activators

- recruit GTFs

- assist RNA polymerase activation

- recruit chromatin remodeling complexes

putting it all together: gene expression process

1. an activator binds to an enhancer located in an NFR

2. activator recruits a chromatin-remodeling complex and HAT to the NFR. nucleosomes may be moved, histones may be evicted, some histones are subjected to histone modification, and the core promoter sequence is revealed

3. GTFs and RNA polymerase II bind to the core promoter to form the pre-initiation complex

4. elongation: histones are covalently modified by acetylation and evicted or partially displaced. behind the open complex, histones are deacetylated and are tightly bound to DNA. chaperones aid in moving these histones around

naked DNA is ____-

vulnerable to attack and damage, so it is remethylated after transcription occurs to protect it

what is ferritin

20 identical subunits that store iron

ferritin gene regulation: low iron levels

when iron levels are low, iron response protein (IRP) bond to hairpin iron regulatory element (IRE) and inhibits translation of the ferritin protein

ex of translation initiation regulator

ferritin gene expression: high iron levels

iron binds to IRP, causing a conformation change releasing it from the IRE and allowing translation to proceed

- ferritin is produced and iron can be stored

why is regulation of protein expression and activity important and relevant?

widespread deregulation of transcription occurs during oncogenesis

viruses are found in...

all three domains

viruses that infect bacteria specifically are called...

bacteriophages or phages

viruses are...

obligate intracellular parasites

- can persist on their own under permissive conditions but can reproduce only within a host cell using tools from the host

- genomes may code for a few enzymes or proteins, but they must use ATP, AAs, tRNA, nucleotides, ribosomes, etc from host to replicate

what is a virus made of

nucleic acids, sometimes a few enzymes, and a protein coat

what is the protein coat on a virus called

capsid

enveloped vs naked virus

enveloped virus = animal virus surrounded by a biological membrane acquired during release from the host cell (rarer)

naked virus = virus with no envelope, just a capsid

basic shapes that viruses can exist in

- rod-shaped virus (ex. tobacco mosaic virus)

- oval/spherical virus (ex. smallpox virus)

- icosahedron shaped (ex. adenovirus)

- icosahedron with a tail and landing spikes (ex. bacteriophage T4)

nucleic acid genomes of viruses

- typically a few dozen to a few hundred genes

- smallest known is 1700 bases and a few genes

- largest known has over 2 million base pairs

variation of nucleic acid viral genomes

can be...

- DNA or RNA

- single or double stranded

- linear or circular

- single copy or multiple copies (ex homologous chromosomes)

- segmented or non-segmented (ex one long piece of DNA vs segmented chromosomes)

most common type of viral genome is...

linear double stranded DNA virus because this is the most common type of bacterial ggenome

how many bacteria and viruses exist in the world

10^30 bacteria

10^31 viruses

viral replicative cycle: 3 tasks

get in, replicate genetic material, and get out

7 basic steps of viral replicative cycles

1. attachment to specific host cell surface receptors

2. entry of viral nucleic acid into host cell

3. early gene expression

4. replication of viral nucleic acid

5. late gene expression

6. assembly of new virus particles

7. release of new virus particles from the host cell

1. attachment of virus

- virus attaches to specific host cell surface receptors

- specific interaction of capsid protein (or glycoprotein of enveloped virus) with a receptor on host (usually a glycoprotein with its own natural function)

2. entry: transfer of viarl nucleic acid into host cell

- sometimes just the nucleic acid enters the cell, sometimes the entire viral particle enters the cell

- phage usually have to "inject" their nucleic acid through a cell wall, leaving the capsid behind

- animal viruses typically enter by membrane fusion or endocytosis

membrane fusion

virus envelope is made of the same material as the cell's plasma membrane, gets absorbed into the plasma membrane and the rest of the capsid/virus enters the cell

endocytosis

enters as a lysosome/endosome and then escapes before contents turn acidic, releasing viral protein

3. early gene expression of viruses

- production of enzymes needed to replicate viral nucleic acid (and sometimes shut down transcription/replication of host cell/s DNA

- for some viruses, enzymes for replicating viral nucleic acids aren't made by the host cell, so virus has to bring them along

- not all virus types have a defined early gene phase

replication of viral nucleic acid

- new virions must package the original type of nucleic acid

- viruses have established cycles: same thing must occur every time (programmed)

5. late gene expression

production of capsid proteins needed for assembly of viral particles, and enzymes needed for release of virus from host cell

6. assembly of new virus

- new virus particles are assembled from replicated nucleic acid and newly-synthesized capsid proteins

- usually occurs near the plasma membrane

7. release of new virus particles from host cell

- non-enveloped viruses and mot phage burst cell when released (enzyme made by phage lyses cell wall)

- enveloped viruses (most animal viruses) are released by budding (released at regions of membrane modified by viral proteins and surrounds the virus with host membrane so the host doesn't recognize the virus as an intruder)

DNA viruses

- most common DNA viruses are ds-DNA viruses

- best studied are phage T4 (virulent) and lambda (temperate)

virulent phage

follows a lytic pathway, resulting in production of new viral particles and release from the cell

temperate phage

can follow either a lytic pathway or a lysogenic pathway, which results in a period of dormancy that they can exit from at a later time

- if cell conditions are really good or really bad, goes to lytic pathway

- if cells are so/so, remains dormant

replicative cycle of virulent phage T4 in E. coli

1. viral DNA is injected into the cell, leaving the capsid behind

2. early viral mRNA is translated to create two proteins: a nuclease that cuts up host DNA and a protein that modifies RNA pol so it can't bind to host DNA, only to the late viral promoters, which have a diff promoter sequence

3. RNA polymerase is modified and viral DNA is replicated

4. late viral mRNA is translated with the modified RNA pol to make the viral exit particles

5. components are assembled into a virus

6. ~100-200 new virus particles lyse the cell and are released