Viruses and bacteria + The Eukaryotic Genome + Gene Regulation

Dimitri Ivanovski showed that the disease called tobacco mosaic disease was caused by an agent that could pass through what? This helped initially define viruses as what?

A ceramic filter that bacterium won’t fit through, so viruses were defined as a disease agent that’s smaller then a bacterium

Viruses are classified by

• host specificity + pathology of disease they caused

• RNA or DNA + single or double stranded

• by shape (with electron microscope)

What is a Virion?

Virus particles that typically consist of a simple protein coat or capsid + nucleic acid; animal viruses often also have a lipid bilayer

Viruses that infect bacteria (bacteriophage): 2 types & their reproductive cycles

Virulent phage → lytic reproductive cycle

• phage binds to bacteria → injects DNA

• bacteria immediately produces more phage proteins and phage DNA

• When cell used up, cell lyses (disintegrates) and newly assembled viruses release

Temperate phage → lysogenic reproductive cycle

• inserts DNA (called prophage) into bacterial chromosome, waits while bacteria divide (replicating prophage along with its own DNA)

• prophage pops out of chromosome and resumes cycle

Viruses that infect eukaryotes

Many similar to phage except for shape - many RNA/DNA viruses are like lytic phage (infect cells, make self copies, often destroy cell in process)

Retroviruses: bend central dogma rules by making DNA copy from an RNA genome → DNA copy can be integrated into host chromosome and remain there inactive for a while before it starts producing viral particles again

Specialized transduction (genetic material exchange in prokaryotes)

When prophage leaves chromosome, its sometimes sloppy and takes gene from bacterial chromosome with it → bacterial gene gets inserted into the chromosome of another bacteria when it’s infected by the prophage

Generalized transduction (genetic material exchange in prokaryotes)

Sometimes phage do bad job of chopping up bacterial DNA and instead of packaging phage DNA into virons, they package chunks of bacterial DNA.

When virions infect new bacterium, chunck of bacterial DNA gets injected into infected bacterium and can be incorporated into chromosome

This can be used to map genes

Transformation (genetic material exchange in prokaryotes)

Bacteria sometimes takes up DNA floating around in environment - this genomic DNA can be integrated by recombination.

There is a type of non-genomic DNA called plasmids that are small DNA circles that can also be used to transform bacteria → don’t integrate with bacteria but can duplicate through DNA replication

What are R-factors?

Plasmids (small DNA circles that can be used to transform bacteria) that carry antibiotic resistance genes

Conjugation (genetic material exchange in prokaryotes)

E. coli bacteria come in two strains, F+ (male like, have an F-plasmid) and F- (female like). F+ conjugate with F- Bacteria and donate one strand of their F-Plasmid to F- cell, making it F+

• This occurs much more frequently

• F DNA takes some chromosomal DNA with it during conjugation

• Transferred DNA can be incorporated into chromosome of F-cell by recombination

Integrated F strains are called Hfr (high frequency of recombination)

Can see in what order chromosomal genes enter F- bacteria (and therefore map them) by stopping conjugation at different times

Inducible enzymes vs constitutive enzymes in Prokaryotes

Constitutive = always on

Inducible = only turn on genes that code for metabolic proteins that are needed

Lactose is a carbon source that __ (lives in your gut) can use. What 3 proteins are needed to use it? How are the genes for those proteins organized? When are they transcribed?

E. coli can use lactose as a source of carbon but needs the proteins

β-galactosidase, β-galactoside permease, β-galactoside transacetylase

Genes are all in a row and transcribed from the same promoter in 1 big RNA

Only transcribed in response to lactose;

• absence of lactose: protein called the lac-repressor binds to specific DNA sequence called the lac operator → when repressor binds to the operator, it blocks transcription specifically of those genes

• Presence of lactose: lactose binds the repressor → repressor changes conformation + releases from operator → inhibition of transcription is lifted and genes are transcribed

What is an operon

A unit of DNA that’s controlled by a single promoter and who’s transcription is moderated

Exons vs Introns

Exons = -parts of a DNA sequence that code for a protein

Introns = non-coding sequences in eukaryotic genes → removed by a mechanism called splicing (like cutting a scene out of a film)

3 forms of RNA processing

1. Splicing = snRNPs recognize specific sequences at the boundaries of introns and exons → U1 binds to exon-intron boundary; U2 binds to intron-exon boundary, others bind to form a complex that snips out intron and joins the ends of the exon

2. A methylated guanosine cap is added to the 5’ end → helps translation and RNA stability

3. Polyadenylation = 3’ untranslated region is snipped off and replaced with a poly A tail (string of abt 100 adenosine nucleotides), also improving stability

How do telomeres fix the issue of DNA getting shorter every time it replicates (so that genes don’t disapear)

The enzyme telomerase adds short repetitive stretches of DNA, called telomeres, on the ends of chromosomes that don’t code for any gene, providing a buffer between the ends of the chromosome, which are shortening with each cell division, and the DNA encoding for genes

*vast majority of somatic cells don’t make telomerase and just sense when telomeres are getting too short

What are centromeres

repetes of non-coding DNA associated with the centromeric region of the chromosome, possibly involved in binding kinetochore proteins

What are transposable elements (of DNA)

Non-coding DNA that can hop around the genome

Retrotranspons

• LTR

• LINES

• Alu elements

make copies of themselves by being transcribed then using reverse transcriptase to make a DNA copy of the transcript that then re-inserts into the genome

• LTR retrotranspons look like retroviruses + encode retroviral proteins but can’t leave the cell bc they can’t make functional coat proteins

• LINES (non-LTR) don’t look like retroviral proteins but do encode reverse transcriptase and an endonuclease that lets them insert into the genome

• Alu elements (mutated version of functional 7SL RNA gene) don’t encode proteins; rely on reverse transcriptase from other elements to allow them to jump

DNA transposons

Splice themselves out of the genome then jump back in by enxoding a special enzyme called a transposase - these genes appear to be DNA parasites with no useful purpose and only replicate bc they can

Pseudogenes

Sometimes mRNAs get copied into DNA and inserted into genome, making processed pseudogenes

OR

Chromosomal duplicatoin produces multiple copies of a gene, some of which are eventually inactivated by mutation, producing a non-functional pseudogene

Ribozymes

RNA enzymes that can catalyze a variety of reactions just like proteins

Self-splicing introns

introns that splice themselves out - stretch of RNA is able to fold into a structure that has catalytic activity in order to to splice themselves out of RNA

Housekeeping genes

The cells necessary for basic functions of metabolism, transcription, translation, etc

What is the main way eukaryotic cells control what proteins will be present

They control the transcription process (transcription of DNA to mRNA) by regulating the ability of RNA polymerase to initiate transcription → this is determined by the promotor element (usually a TATA about 25 bp upstream) and a few other types of regulater sequences 50-150bp upstream

3 polymerases in eukaryotes

Pol # transcribes __

Pol 1 → rRNA

Pol 2 → mRNA

Pol 3 → tRNA

In Eukaryotes, polymerase doesnt just bind DNA and transcribe - what other proteins need to “prepare the way”?

proteins in the TF2 (transcrpitoin factor for Pol II) family - the main one is TFIID, a complex of proteins including 1 TBP that binds the TATA box DNA; TFIID recruits other TFs that recrute even more TFs that eventually recruit RNA Polymerase II

2 types of DNA elements that control transcription in eukaryotes

Enhancers (bind activators with RNA pol) and Silencers (bind repressors) - can act at a great distance (like 20000 bp away in any direction), and can act in either orientation (can invert the element and it will still work)

• Theorized that DNA folds and enhancer binding proteins contact polymerase that way (needs to fold bc they are so far apart)

both bind specific sequences by having protein domains that probe the major groove of the DNA (wide groove)

What are nucleosomes? Do they inhibit transcription? Heterochromatin vs euchromatin

DNA wrapped around histones - inhibit binding of some transcription factors and therefore transcription (unknown mechanism)

heterochromatin (tightly bond DNA; stains darkly) is not transcribed in contrast to euchromatin (lightly stained)

X + Y Chromosomes → effect of condensing

• Barr body

Females inactivate one X chromosome by condensing it into a state that prevents transcription if Y chromosome occurs; Phenotype affected by what X chromosome is condensed

Barr body = inactivated chromosome

Gene Amplification

Controlling transcription rate by having multiple copies of the same gene

Alternative splicing

Sometimes when splicing intron out of an RNA, an exon will be spliced out as well making a protein thats missing a chunk → presence or absence affects protein functions

EX: by creating mutations in fruit flies that affect which splice variant is made, you can create transgendered flies

Transcription regulation: RNA stability

RNA must be unstable otherwise theres no point to regulating transcription - The 3' and 5' untranslated regions of a transcript affect stability and therefore accumulation of a particular transcript

Control of Translation: RNA interference

occurs when cell makes small interfering RNAs that don’t encode a protein but are complementary to the mRNA of a gene → with the help of protein Dicer, these siRNAs bind to complementary RNA and block its translation

Post - translational controls: Phosphorylation

Addition of a phosphate group to a protein

Enzymes that phosphylate other proteins = Kinases

Post - translational controls: Selective protein degradation

Degradation = protein gets destroyed

Proteins marked by ubiquitination signifies they are destined for degradation

Tradeoffs of regulating transcription vs regulating proteins post translation

Regulating transcription is efficient bc no energy is expended making unused proteins, but it’s slower because needed proteins have to get made
Post-translation regulation is faster but involves making proteins that do nothing while waiting to be activated (or destroyed)