Exam 2 BIO457

What are solitary genes?

genes that are only represented once in the haploid genome

Why do plants have more chromosomal DNA than humans if they aren’t as complex?

they have a different organization of genes

what are genes that belong to families of 2 or more similar genes called?

duplicated protein-coding gene

gene families description

duplicated genes encoding proteins with similar AA sequences

describe a protein family

encoded closely related proteins

what are sequences in gene families that don’t code for proteins but very similar in seq to the funcitnoal genes called?

pseudogenes

why could pseudogenes be inactive?

• they lack proper signals/sequences for transcriptional initiation (lacks a promoter or enhancers)

• improper splicing at junctions

• prematurely terminate translation

what examples are tandemly repeated genes

genes for rRNAs, tRNAs, and histone proteins

What are tandemly repeated genes? Why do we have them?

multiple repeated genes encode almost identical/identical proteins or functional RNAs; copies of a sequence appear one after another over long stretch of dna

• We need to make a ton of rRNAs, tRNAs for translation

how is DNA organized in prokaryotes vs eukaryotes

prokaryotes: one circular chromosome

eukaryotes: many chromosomes compacted in nucleus as chromotin with histone proteins

What are the 5 major types of histone proteins

H1, H2A, H2B, H3, H4

describe chromatin in extended form

DNA is in nucleosomes (bead-like structures) that have linker DNA between them; 146 bp of DNA wrap around the octamer core (2 each of H2A, H2B, H3, and H4)

What is the primary structural unit of chromatin?

nucleosomes

describe the condensed form of chromatin

looks like 30 nm diameter fibers, where 2 strands of nucleosomes are stacked and packed into a zig-zag ribbon that is wound into a helix

• has 6 nucleosomes per turn

• solenoid shape

• H1 is bound to the linker region between nucleosomes to help fold the structure up

what does chromatin do to form chromsomes?

supercoiling and interaction with non-histone proteins

What happens to do chromosomes look like when they have histones depleted?

long loops of DNA are seen to emanate from a central x-shaped chromosomal scaffold

when is DNA in chromosome form vs spagetti form

interphase- doesn’t need to be condensed

need condensed form during prophase, metaphase, etc.

what protein is a major component of the chromosomal scaffold? what is its function

topoisomerase II—> acts as glue to attach DNAs from their SARs (scaffold-associated regions) to the chromosome scaffold

where are SARs found in DNA?

between transcription units

where are genes in the protein scaffold? what does the scaffold/SARs allow?

chromatin uncoils in the transcribed region between SARs (loops); puts solitary genes closer together and SARs acts as insulators for gene families

What are Smc proteins?

structural maintenance of chromosome proteins for maintaining structure of interphase/metaphase chromosomes

describe Smc structures/interactions

they have 3 domains and bind with kleisin to form a ring-like structure called condensin

• made of 2 monomers (Smc2 and Smc4) that are coiled--coils, associating through hinge domains

• their head domains are linked by kleisin to keep the ring like form

What is condensin?

made of Smc and kleisin to from a ring and hold 2 chromatin fibers together

What 3 functional elements must chromosomes contain

1) replication origins

2) centromeres

3) telomeres

What are centromeres and what proteins help them?

short, AT rich DNA sequences that serve as sites for binding spindle fibers

• CENP- A,B,C,D proteins

What are telomeres and what happens to them during dna replication?

the telomeric repeats rich with G’s at the ends of DNA that folds back on itself through G-G pairing to block the end and stabilize the DNA; get shorter each time the DNA is replicated

What happens if there are no telomeres?

the chromosome is highly sensitive to enzymatic degradation and can easily interact w/other chromosomes

What is telomerase and its function?

it adds cap and is actually a complex of both protein+RNA (internal RNA template helps direct DNA synthesis of telomeric repeats by having complementary RNA seq)

how do telomeres get shorter?

• lagging strand of DNA replication has a primer removed at the 5’ end, and can’t be filled back in

• telomerase recognizes the 3’ of the parental strand and adds >100 repeats of 5-12 G-rich sequences to it

• THEN, primase/poly-alpha proceeds laying down primers/making more Okazaki fragments for the lagging strand until telomerase recognizes once enough repeats are added

What happens as our cell start to age in regards to DNA?

DNA starts to shorten—> telomeres act as a timing mechanism, so that cells can only divide a certain number of times (~50) before they undergo apoptosis (Hayflick limit)

old cells might have more mutations, so telomerase stops functioning after we’re born so that the biological clock can start

What happens to cause cancer/malignant cells regarding dna/telomerase?

Cells that have undergone mutations in their divisions—> telomerase is reactivated, allowing cells to divide uncontrollably and biological clock is turned off

what does gene expression mean?

the entire process where information encoded in a particular gene is decoded into a protein

what is teh most important control point in determing gene expression adn how much mRNAs/proteins are produced?

decision to initiate transcription

difference between the purposes of gene regulation in single-celled versus multicellular organisms:

prokaryotes: gene control allows rapid adjustment to environmental changes so growth and division is optimized—>

• most of the regulatory controls are easily reversible systems and allow rapid, short-term switches from one biochem pathway to another

eukaryotes: purpose of gene regulation is to control genetic program underlying development and differentiation

• precise decision so that the right gene is activated in right cell at right time during development of all the different cell types that make up an organism

What mechanisms does regulation provide for cells about gene expression

determining kinds and amounts of all cellular molecules (proteins) produced and times/locations of their synthesis

what does regulation of gene expression underlie?

• differentiation (how cell know what to become) 

- cells respond and adjust to stimuli and environmental changes

What are the 7components of regulatory proteins/transcritpion factors?

1) found in the nucleus

2) act by universal mechanisms bc DNA is DNA

3) composed of modular components (domains)

4)proteins are modified

5) proteins are bound to promoters/enhancers

6) cooperative reguation by multiple proteins

7) genes are controlled as networks to link response of genes

describe how regulatory proteins/TFs are composed of modular components

• DNA binding domain (spec seq)

• protein binding domain (to form complexes)

• transcriptional activation domain (reserved for general factors)

describe the most common forms of regulatory protein modification

• phosphorylation (can activate proteins by causing a conformational change into their functional form)

• binding of another molecules/ligands (hormones, GF, etcc) to a specific domain (causes conformational change to become functional)

• addition of another subunit to introduce a functional domain

• removal of inhibitor

describe how regulatory proteins binding to promoters/enhcancers affects transcirption

• it helps assemble general TFs/loop DNA to interact better with RNA poly

describe cooperative regulation by regulation proteins

• certain proteins can be part of both complexes that activate/repress transcription (has different domains)

• gene control requires both common and cell-specific transcription factors

  • bc very different cells have both identical and different needs for proteins/gene activation

  • different combinations of them can have more specificity

describe how genes are controlled as networks and why

• multiple genes recognized by group of specific TFs, linking their response

  • the TFs bind to a common enhancer sequence that are associated with all the genes (common enhancer)

• it makes sure all protein elements of a full quaternary protein are made, since they are usually solitary genes

what are examples of common enhancers

response element, octamer sequence, heat shock element

what are other mechanisms to control transcription other than TFs?

packaging of DNA/associated modificaitons, chemical modification histones, direct modificaiton of DNA

describe how packaging of DNA into chromatin and associated modifications regulates genes

• condensed DNA is a barrier to transcription—> gene is off

• chromatin opens up to expose individual nucleosomes that general TFs and RNA poly II during transcription

how are histones modified

acetylation, methylation, phosphorylation, ubiquitinylation

• modified on their flexible N-terminus histone tail

• tail is required for condensing DNA by associating with/wrapping around the DNA

describe histones are methylated

on lysines; often these sites are associated phosphorylation and acetylation sites

describe how histones acetylated

most common modification of all histone proteins; on lysines

describe histone ubiquitination

marks proteins for degradation (on H2A and H2B)

describe acetylation/deacetylation of histone tails

• this occurs on the lysines, since they act as a base/have a positive charge and DNA is negative —> neutralizes the charge to break DNA interaction and interaction with other histones

• causes partial unwinding of DNA and breakdown of octamer core—> allows TFs to have access

• done my histone acetyltransferase (HAT)

• deacetylase (HDAC) removes the groups to tighten chromatin/repress transcription

how is acetylation activity involved in some cancer cells

overexpressed/sustained HDAC activity in cancer cells allows them to turn off genes associated with apoptosis

what modification prevents acetylation? why is this done?

methylation of lysines; to keep certain genes turned off

describe the direct modificaiton of DNA

methylation of cytosines by cytosine-5-methyltransferase

• found in control regions like promoters/enhancers to block access of TFs

• this turns genes off (heterochromatin)

• usually done to the “junk” dna

there is _________ between TFs and sequence regonition

synergy

how are bacterial genes organized and why?

in operons, where genes for related proteins are put togehter (polycistronic mRNA)

• allow fast transcription/tranlsation of related genes since bacteria need to be able to rpaidly change to their environment

what is the operator sequence and where is it found?

a sequence in the promoter region that is bound/affected by repressors/activators

affects of activators vs repressors

o   repressors adjust the rate of initiation downward from higher base level

§  repressor-regulated operons are characterized by increased transcription when free of repressor and reduced rate when bound to promoter/operons

o   activators adjust promoters upward from lower base level

§  activator-regulated operons are characterized by very low or nonexistent transcription rates when free of the activators/higher rate when they’re presen

what allows RNA polymerase to bind to promotors and acts as an activitor

sigma factors

Describe the lac operon’s function/'composition

has 3 genes producing 3 enzymes for breaking down lactose in E.coli

describe what the lac operon is repressed by and when

lac repressor

• made from separate gene from lac operator as a tetramer in its active form

• binds the operator seq in it DNA binding domain to inhibit transcription, by preventing RNA poly binding

• repressor binds when no lactose is present, since no lactose needs to be broken down

• also has binding site that recognizes/binds to allolactose —>causes conformational change that reduces affinity with operator, causing repressor to release and transcription to occur

describe the purpose of the trp operon

produces enzymes needed for tryptophan synthesis

describe how the trp operon is regulated

the operon is normally turned on a lot, but can be reduced when there is a buildup of trp

• trp repressor has 2 binding sites: for operator and for tryptophan

  • made in inactive form, where it has no affinity for operator

• if tryptophan become available/builds up, excess trp binds to repressor, causing conformational change to repressor so it can bind to operator

  • trp acts as a corepressor

describe the structure of the lac repressor

• 4 polypeptides (tetramer)

  • tetramer contains helix-turn-helix motifs (alpha-helices), that fit into major grooves in DNA

what structure do most repressors take on?

homodimers (dimers of single polypep) that have alpha helices

describe regulation of repressors in bacteria and why it occurs

they are also coded for in operons, which are autoregulated-(negatively controlled by their own products, when products bind to their operator)

• ensures that there are always some repressor molecules so the cell can rapidly respond to changes

what kind of operons do activators usually control

those encoding enzymes that carry out major functions (transport, response to stress/DNA damage)

describe regulation of activators

they are also autoregulated and lac/gal/ara operons have positive regulators

• catabolite activator protein (CAP) or cAMP receptor protein (CRP)

  • glucose is use first as energy source in prokaryotes, and cells turn to other sugars if not there

  • CAP is made in inactive form, but when glucose is low, ATP is converted to cAMP—> cAMP binds to CAP to induce a conformational change that activates CAP

  • active cAMP-CAP molecule binds to CAP site of the lac, gal, and ara operons simulataneously and then binds to RNA poly-sigma 70 to transcribe

  • allows networking of lac/gal/ara genes

describe the structure of transcirption factors

have modular components of different domains (DNA-binding/protein binding domains)

• alpha helices to bind DNA major groove

• classified according to type of DNA binding domain

What are the 3 common classes of TFs?

zinc-finger proteins, leucine zippers, and helix-loop-helix proteins

What are the 3 classes of zinc finger proteins?

1) C₂H₂ zinc finger

2)C₄ zinc finger

3)C₆ zinc finger

describe the C2H2 zinc finger

class 1

• contains repeating consensus sequences that bind 1 Zn2+ ion to 2 cysteines and 2 histidines

• usually have >3 of the units wrapping around a DNA helix

• bind as monomers

describe zinc-finger proteins in general

• stretch of amino acids that are folded around a central Zn²⁺ to produce compact domain from a short length of peptide

describe the C4 zinc finger protein

class 2

• consensus seq that has 2 groups of 4 cysteines, each binding a zinc (2 fingers)

• alpha helices regions bind the DNA

• is similar to C2H2, but generally has >2 fingers and binds has homo/heterodimers rather than monomers

describe C6 zinc fingers

class 3

• has consensus sequence with 6 cysteins that bind 1 Zn and folds regions into a compact globular domain (fist)

• binds DNA as a homodimer

describe leucine zippers; what is a more general name for them?

• every 7th AA in the C-terminal end, there is a hydrophobic leucine, causing dimerization (homo/hetero) through a zipper like interaction between amphipathic alpha helices

• can bind DNA as dimers through the 2 alpha helices at the N-terminal end that grip the dna like a pair of scissors at adjacent grooves

  • these helices contain basic (postiviely charged) amino acids (K,R,H) that can interact due to the charge difference

bZip (basic zippers)—> have other hydrophobic AA other than leucine

describe helix-loop-helix proteinns

• forms homo/hetero dimers through interactions between its amphipathic alpha-helical regions

• has 2 alpha helical regions (many conserved hydrophobic residues for dimerization) separated by a nonhelical loop

• most have a highly basic region to bind DNA at the N-terminal end —> called bHLH proteins

  • class A: ubiquitously expressed (in all cells)

  • class B: expressed in tissue-specific manner

what are the reasons that formation of heterodimers are important

1) allows increases in potential DNA sequences it can bind due to different combinations of monomers

2) allows different combinations of activation domains to be brought together at regulatory sequences, allowing cooperative regulation/protein-protein interactions

• allows different functions regarding DNA binding/transcriptional regulation

what are enhanceosomes?

large complexes of TFs that bind to enhancer sequences; form due to their activiation comains

• has 2 heterodimers and 4 monomers