Cell Biology Exam 1 Review (Chps 1-4)

Define cytoskeleton and describe its functions

• Network of different protein fibers that provide many functions

• Functions:

  • Adopt different shapes

  • Organize organelles in specific positions

  • Interact with the environment

  • Carry out directed movement (cytoplasm and vesicle within cell)

  • Replicate

  • Support volume of cytoplasm

What are the three major types of protein filaments?

• Microtubule:

  • Hollow cylinders of protein tubulin

  • Largest

• Intermediate filament:

  • Rope-like fibers of intermediate filament proteins

• Actin/microfilament:

  • Helical polymers of protein actin

  • Smallest

Describe the characteristics of Intermediate filaments

• Forms mesh like network in cells; provide tensile strength

• Type of cytoskeleton that makes up nuclear envelope

• Monomers twist together to form “rope-like” polymer

• Ex.) keratin and lamins

Do intermediate filaments have a role in cell movement?

No, main function is structural where tensile strength enables cells to withstand mechanical stress

What role do intermediate filaments play in epithelial cells?

• Skin cells have high concentration of keratin

• Intermediate filaments from one cell interact w/ others from adjacent cells via desmosomes

• Desmosomes:

  • cell-cell junction that joins neighboring cells together

How do intermediate filaments protect cells from mechanical stress?

• Intermediate filaments prevent the rupture of cells, due to desmosomes, allowing the cell to remain intact and together

Describe the intermediate filament structure

• Rope-like proteins

• Monomers: globular ends w/ long a-helical region form dimers

• Dimers twist together to form strong multimers that are antiparallel

• Rope-like filament made of tetramer that are packed into a helical array of 8 tetramer strands

• Globular ends exposed to allow interaction w/ proteins

What can mutations in keratin genes lead to and why?

• Epidermolysis bullosa simplex (EBS):

  • rare genetic disorder caused by defects in gene coding for keratin proteins

  • Causes extremely sensitive skin, blisters, and skin to breakdown easily

• Why?

  • mutation leads to loss of function in keratins causing the inability of cell to maintain structure under pressure

How would intermediate filaments be categorized?

• Cytoplasmic

  • Keratins (in epithelia):

    • Diverse every kind of epithelia in body

  • Neurofilaments:

    • in nerve cells

• Nuclear

  • Nuclear lamins:

    • in all animal cells

    • strengthen the nuclear envelope

Describe Lamins

• Type of intermediate filament that makes up structural elements of nuclear lamina

• Essential in maintaining its structure

• Plays an important role in cellular functions, mitosis, where nuclear envelope breaks down during prophase and reforms in telophase

What are results of defects in nuclear lamin proteins?

• Progeria:

  • premature aging causing irregular-shaped nuclear envelopes

  • potentially associated with defects in mitosis, causing unstable cell division

What are characteristics of microtubules?

• Associated with motor proteins

• Thickest type of cytoskeleton

• Made of tubulin monomers

• Pulls chromosomes apart during anaphase

• Involved in vesicle trafficking/ movement

Describe microtubules and their cellular functions

• Hollow, tube-like filaments

• polymer of tubulin

• extend from microtubule organizing centers (MTOC), like centrosomes, spindle poles, and basal bodies

• Cellular functions:

  • Spindle formation

  • Cilia/flagella movement

  • Intracellular transport

Describe the structure of microtubules

• aB dimers arranged/stacked together in the pro filament oriented in the same direction

• 13 parallel pro filament has a structural polarity; one need B-tubulin (plus end) and a-tubulin ( minus end)

• Pro filaments make up “hollow tube”

• Dimers add to plus end faster than minus end

• Primary structural elements: flagella, cilia, centrioles

Define dynamic instability

• Microtubules rapidly extend (polymerization) and shrink (depolymerization)

• Grows outward from an organizing center by the addition of aB tubulin dimmers to end

• Crucial for rapid remodeling

• GTP hydrolysis controls dynamic instability and dimers can hydrolyze GTP

What is one example of dynamic instability?

• movement of chromosomes to opposite poles of a dividing cell during mitosis called spindle fibers

Describe the guide transport of microtubules

• Many cells are polarized (microtubules extend in one direction)

• Example: neurons

  • Outward transport:

    • vesicles with membrane or secretory proteins

  • Inward transport:

    • Damaged proteins and organelles

Define motor proteins and the two types in microtubules

• Motor proteins:

  • move along the tracks in a specific direction

• Types:

  • Kinesin:

    • walk along microtubules and involved in vesicle transport

    • plus-end directed ( outward transport- away from cell body)

    • movement from “-” to “+” end of microtubule using energy fro. hydrolysis of ATP

  • Dyenin:

    • minus end directed ( inward transport)

    • movement from “+” to “-” end of microtubule filament towards cell center

    • It converts chemical energy from ATP hydrolysis into mechanical energy of movement to walk along microtubule while carrying a vesicle

What are the characteristics of Actin/ microfilaments?

• The thinnest type of cytoskeleton

• Involved in cell crawling

• Associated with motor proteins

• made of actin monomers

• Involved in vesicle trafficking/ movement

Describe Actin/ microfilaments and examples

• One of the most abundant proteins in eukaryotic cells

• 5% of total cellular protein

• Polymers twist into helix

• Actin monomers can add to plus or minus end, but add to plus end faster

• Actin:

  • ATP →tightly bind to filament

  • ADP → not as tightly bound

• Dynamic instability:

  • rapid extension and shrinking of filaments

Describe the type of motor protein in actin/ microfilaments

• Myosin:

  • Found in most cells and used in vesicle transport

  • used to move actin filaments relative to plasma membrane (cell shape change)

  • ATP dependent

  • Plus-end directed motor protein (- to + end)

  • movement uses energy of ATP hydrolysis to provide energy for changes needed for movement

Do all cells have the same DNA, and how do we get different cells?

• Yes, most cells have the same DNA, but they become different through gene expression, where cells turn certain genes on or off

List the 4 nucleotides and how they interact with each other in the double helix

• Adenine, Thymine, Guanine, Cytosine

• A → T ; G → C

Define a gene

• Linear sequence of nucleotides along a segment of DNA that provides coded instructions for synthesis of RNA, then translated into protein, leading to the expression of hereditary character

Describe gene expression

• The process of turning on a gene to code for mRNA to produce protein

• It must be regulated, because not al genes can be turned on or off at the same time

• Regulation of gene expression conserves energy

Describe the coding region of DNA

• The genetic doe of how DNA codes for an amino acid is nor obvious from a base pair sequence

• There are protein coding region soft DNA where the DNA is used to make RNA and then protein

Describe the non-coding region of DNA

• ~2-5% of the human genome contains genes

• Remaining portion= non coding DNA (do not code for protein)

• Role: not fully understood, but may be important in regulation of gene expression

• Genome: all DNA/ genetic information

Compare prokaryotic and eukaryotic genomes

• Prokaryotic:

  • genomes are circular and contain plasma membrane and chromosome

• Eukaryotic:

  • Linear and organized into multiple chromosomes

  • Chromatin:

    • DNA complexed with proteins

  • Chromosomes:

    • linear DNA molecules and associated proteins that is folded into a compact structure

  • Chromatin condenses to form chromosomes

Describe eukaryotic chromosomes

• Except germ cells (sperm and eggs) and RBCs, human cognation two copies of each chromosome

• Maternal and paternal pairs —> homologous chromosome

• only non-homologous pairs are sex chromosomes

Define karyotype

• Ordered display of the full set of 46 chromosomes (23 pairs)

Describe the types of chromatin

• Euchromatin:

  • more expanded DNA, less condensed

• Heterochromatin:

  • Highly condensed DNA, more DNA packaging

  • more likely to see in dividing cells

For gene expression differences, do you think that it is all or nothing?

• No, generally a mixture of euchromatin and heterochromatin

When does DNA replication occur?

• Occurs during interphase

• duplicated in preparation for mitosis

Describe the anatomy of a chromosome

• Duplicated mitotic chromosome is highly condensed

• Contains two identical daughter DNA molecules

• Each one chromatid (sister chromatids)

• Centromere:

  • specific sequence that allows duplicated chromosomes to be separated

Describe interphase vs m-phase chromosomes

• Interphase:

  • when chromosomes are duplicated

  • chromatin condenses to prepare for distribution

• M-phase:

  • mitosis leading to distributed 2 daughter cells

Define a nucleosome

• Structure when DNA wraps around proteins called histones

Define a histone

• Proteins that bind DNA for packaging

Describe the anatomy of a nucleosome

• Beads on a string

• String = linker DNA

• Beads = nucleosome core:

  • DNA wrapped around proteins called histones

What properties would histones have to bind DNA?

• Positively charged histone subunits

Describe the process of nucleosome anatomy

• Histones from an octamer (8 monomers) w/ 2 of each histone: H2A, H2B, H3, and H4 all joined together in a cylindrical structure with a positively charged amino acid to interact w/ DNA and form a nucleosome

What is the importance of H1?

• Histone H1 binds to the linker DNA and bends it, so that those nucleosomes can twist into a helix or spiral

• has a long C-terminal that helps bind to chromatin

Describe DNA packaging from least to most condensed

• Naked DNA/DNA double helix (least)

• Beads on string (nucleosome)

• 30-nm filament

• Radical loops

• Condensed section of chromosomes

• Mitotic chromosome (most)

Why would we need to access tightly packaged DNA?

• DNA replication

• DNA repair

• Gene expression ( transcription and translation)

What is the role of the H1 linker protein?

• Pulls nucleosomes together and pack them tightly to help bind the linker DNA, which aids in chromatin condensation

Describe the role of histones H2A, H2B, H3, and H4

• H2A and H2B provide structural, disk shaped core that is the fundamental unit of chromatin

• H3 and H4 are essential for initial tetramer formation

Describe whether euchromatin or heterochromatin can result in increased gene expression and why?

• Euchromatin can result in increased gene expression b/c its less condensed and loosely coiled, meaning more accessible for transcription

Describe ATP-Dependent Chromatin Remodeling:

• Regulates DNA condensation

• Adjust the structure of chromatin to allow access to proteins involved in DNA replication and repair

• Adjusts position of DNA wrapped around nucleosomes

• Uses energy from ATP hydrolysis and inactivated during mitosis

What are the effects of post transitional modifications to histones?

• Addition of these chemical groups to stones cause changes in gene expression

• Alter chromatin structure based on chemical modification of histones (could reduce strength of interactions for histones)

• Can serve as binding or docking sites for proteins that alter condensation of DNA

Describe the Central Dogma of Biology

• DNA is transcribed to RNA, then translated to proteins

Does this process of Central Dogma ever of backwards?

• No, but retroviruses like HIV are an exception b/c it has a reverse transcriptase enzyme that allows this reverse

Describe Transcription

• One strand of DNA serves template (3’ to 5’)

• Coding strand: matches RNA

• RNA added in 5’ to 3’ direction

Describe Huntington’s Disease (HD):

• Fatal genetic dominant disorder that causes progressive breakdown of nerve cells in brain cell causing loss of motor and memory

• Known as the quintessential disease where every child of a parent w/ HD has 50/50 chance of carrying gene

• Happens when patients have a mutation in the Huntington gene, resulting in production of rom of Huntington protein that attacks neuron

Compare RNA and DNA

• RNA:

  • single stranded

  • ribose nucleic acid w/ 2 OHs attached

  • uses Uracil instead of Thymine

  • RNA nucleotides:

    • purines → A and G

    • pyrimidines → C and U

• DNA:

  • Double stranded

  • deoxyribose nucleic acid with 1 OH

  • uses Thymine

Describe the roles of the types of RNA

• mRNA:

  • code for proteins

• rRNA:

  • form the core of the ribosome and catalyze protein synthesis

• miRNA:

  • regulat gene expression

• tRNA:

  • serve as adaptors between mRNA and amino acids during protein synthesis

What can all cells do and name an example

• can express different genes at different rates

• The same cell can make a lot of gene A and at the same time make a smaller amount of gene B

• Ex.)

  • Skin cells need some DNA polymerase, but not all the time

  • Insulin secretion in the pancreas

What is needed for Transcription?

• Template:

  • region of DNA to be transcribed into RNA

• Monomers for new RNA strand:

  • ribonucleotides (ATP, UTP, CTP, GTP)

• Enzymes to polymerize monomers:

  • RNA polymerase

• Key differences to DNA replication:

  • only one strand of RNA made and no primers needed

Why is the term “pre-mRNA “ used when genes are transcribed here?

• Because splicing addition of 5’ caping, and adding of poly- A - tail needed for mature RNA

What are the basics of Transcription?

• RNA polymerase moves along DNA by unwinding the DNA helix in front

• Add ribonucleotides one by one to RNA chain and uses DNA chain as template

• Resulting RNA transcript is complementary to template

• Polymerase moves in 3’ to 5’ direction and displaces newly formed RNA strand along DNA template

Describe the type of RNA polymerase in Eukaryotic cells

• RNA polymerase I:

  • makes most rRNA genes

• RNA polymerase II:

  • makes mRNA, miRNA, and small RNBAs in spliceosomes

• RNA polymerase III:

  • makes tRNA, Ss rRNA, and other small RNAs

Describe the starting and stopping transcription process

• Promoter:

  • region of DNA that signals the start of RNA synthesis

• Terminator:

  • region of DNA that signals the end of RNA synthesis

Process:

• RNA polymerase encounters DNA nd slides down the double helix and latches on tightly to promoter

• Goes on to open up double helix to allow the process to continue until reaching the terminator region

• the polymerase halts and releases the DNA template band the new RNA transcript

• Transcription moves in 5’ to 3’ direction of the new mRNA transcript (left to right)

What are the parts of Transcription?

• Template strand:

  • DNA strand being transcribed

• Promoter:

  • TATAA Box in eukaryotic cells. Transcription factors and RNA pol II assemble at promoter region on non-template strand

• +1 or start site:

  • actual starting point of transcription located on template strand

• Upstream:

  • sequence before start site

• Downstream:

  • sequence after start site

• Coding strand:

  • contains promoter region (TATAA Box) orientation of promoter determines which direction that gene is transcribed

Define Transcription factors and describe the factors necessary to promote initiation process of Transcription of agent in eukaryotes

• Transcription factors:

  • proteins that bind to DNA to facilitate transcription

  • these factors assemble at promoter to initiate transcription

• Process:

  • TATA binding protein (TBP) recognizes and binds the core promoter (TATA box) and bends DNA

  • TFIIA and TFIIB join and TFIIB determines start site

  • TFFIIF bonded to RNA pol II brings enzyme to promoter

  • TFIIE and TFIIH bind

  • TFIIH acts as helicase to allow polymerase to start transcribing and phosphorylates RNA pol II tail, which releases general transcription factors (except TFIID)

  • Now RNA pol II adds ribonucleotides (UTP, ATP, CTP, GTP) to growing RNA strand

  • RNA pol II is released to undergo elongation process, where it adds ribonucleotides to the growing RNA strand

Describe the Elongation process in Transcription

• RNA pol II adds ribonucleotides to growing RNA strand, making it longer

Describe the Termination process in transcription

• Termination sequence exists that releases mRNA

• RNA pol II has no specific signals that terminate transcription

• A protein complex will bind to two locations on the growing pre-mRNA once RNA polymerase transcribed

• Complex binds common AAUAAA sequence and a UG sequence

• Protein in complex (CPSF) will cleave pre-mRNA at site between two bound sequences

• This releases the pre mRNA

• Poly (A) polymerase is a part of the same complex and will begin to add a poly-A tail to pre-mRNA (mRNA processing)

Explain the purpose of the three important modifications to eukaryotic pre- mRNA

1) 5’capping:

• essential for ribosome recognition and translation

2) Addition of poly A tail:

• plays a role in nuclear export of the mRNA and potential role in the protection from nucleases

3) Splicing:

• Remove introns (noncoding regions) and link exons (coding sequences) to create mature mRNA

Define Spliceosome

• a large RNA/protein complex

• consists of snRNPs (small nuclear ribonucleoprotein particles)

• snRNPs recognize those specific sequences and catalyze the covalent linkage of exon sequences

• A -branch point:

  • where the beginning of intron is attached to form lariat structure

Describe Alternative Splicing

• 1 gene DOES NOT equal 1 protein

• can produce various mRNAs from pre-RNAs to produce various mRNAs and proteins

• Alternate splicing → rearrangement of protein domains (combination of new exons)→ new “patchwork” proteins

Define Untranslated Region (UTR)

• when not all mRNA transcript codes for proteins

What is a protein domain?

• a segment of a gene codes for a section of a protein that does a specific function/ task

What are the components of a fully processed mRNA transcript ready for export?

• 5’ cap

• 5’ UTR

• Coding sequence

• 3’UTR

• poly-A tail

What are recognized by Nuclear Export proteins?

• 5’cap and poly A tail

• binding both of these act as a signal to exit the nuclear core

• then exchange that export factor for an initiation factor, so the ribosome starts translating

What therapy could treat HD at the level of RNA to prevent the production of toxic protein?

Gene silencing/ Antisense Therapy:

• take known sequence of the HTT gene, generate an oligonucleotide that attaches to mRNA

• Double stranded mRNA will be chopped up b/c it is not recognized/ doesn’t belong

What is translation?

• Using three nucleotides (codon) to code for amino acids

What are some important codons to know?

• Start codon:

  • AUG (Met)

• Stop codons:

  • UAA, UAG, UGA

What are key characteristics for genetic code?

1) Degeneracy:

• amino acids associated with more than one codon

• Ex.) Proline (Pro)

2) Specificity:

• each codon only codes for one amino acid

• Ex.) UGU → always cysteine

What is a wobble nucleotide?

• usually the third nucleotide, because it does not change the amino acid

What are the key parts of Translation?

• mRNA:

  • carries nucleotide code for the protein to be made

• tRNA:

  • molecules interpret nucleotide code

• Ribosome:

  • made of rRNA and protein

  • catalyzes formation of peptide bond

Explain the transfer RNA (tRNA)

• an adaptor molecule made of RNA that interprets the genetic code embedded in mRNA (L shaped)

• Anticodon:

  • sequence on tRNA that is complementary on mRNA

  • must be charged with correct amino acids before use of protein synthesis

Explain Ribosomes

• Facilitate large and small subunits for protein synthesis

• Contains proteins and rRNA (ribosomal RNA)

• called ribozymes b/c contains RNA w/ catalytic activity that does formation of peptide bond

• Found in Rough ER and cytoplasm in eukaryotic

Describe the types of mutations

• Mis-sense mutation:

  • one amino acid change (substitution)

  • Ex.) sickle cell anemia

• Non-sense mutation:

  • mutations result in a premature stop codon, meaning protein never made and mRNA degraded

• Silent mutation:

  • change in mRNA sequence that does not change amino acid sequence

• Frameshift mutation:

  • deletion of one nucleotide of mRNA which results in a change in all future amino acids

  • Most likely a stop codon and truncated protein (never made) or a nonfunctional protein gets degraded

Describe the functions of tRNA binding sites

• “translation sandwich” mRNA between large and small ribosomal subunit

• Aminoacyl site (A-site):

  • binds incoming aminoacyl- tRNA, which carries next amino acid to added chain

  • new tRNAs bind to this site

• Peptidyl site (P-site):

  • where the initiator tRNA first binds (found)

  • holds tRNA molecule attached to growing polypeptide chain

• Exit site (E-site):

  • Binds empty tRNA after amino acid added to chain, then tRNA exits ribosome

Describe the Initiation step of Translation

1) The initiator tRNA and initiation factors recognize 5’ cap and bind to small subunit of ribosome

2) The tRNA small ribosomal subunit bind to 5’ end of mRNA, moves along 5’ to 3’ in search of start codon (AUG)

3) When the start codon is found, initiation factors are related and the large ribosomal subunit binds

Describe the Elongation step of Translation

1) The charged tRNA carrying next amino acid binds to the A site creating new tRNA binding

2) A peptide bond is formed by the uncoupling of the tRNA on carboxyl end of the amino acid attached to the P-site, forming the peptide bond on the A site of tRNA

3) The large ribosomal subunit translocates three nucleotides (shifts down a codon)

4) The small ribosomal subunit moves 3 nucleotides to match the large subunit, and tRNA is ejected

This cycle is repeated until Stop codon is released

In elongation, how does the ribosome know which tRNA needs to be in the A-site?

• Codon in the A-site is a complement for the anti-codon, so the anti- codon and codon match up to bring the amino acid that was attached to the tRNA

Describe what a “charged” tRNA is and describe what enzyme is responsible for the recycling of a tRNA that is “uncharged”

• A charged tRNA means that the tRNA is covalently bonded to its corresponding amino acid

• The aminoacyl tRNA synthetase enzyme recharges the empty tRNA, in order to make more proteins

Describe the Termination step of Translation

1) The strip codons (UGA, UAA, UAG) are not recognized by a tRNA and do not specify an amino acid

2) The release factors ( made of proteins) bind to any stop codon that reaches A-site ( stimulates the release and dissociation of the ribosome)

3) The binding of release factors alter the enzyme activity in the ribosome, resulting in hydrolysis of polypeptide chain from the last tRNA

4) The disassembly of the entire complex occurs after the release of polypeptide chain

5) The ribosome also releases mRNA, that is later degraded, and dissociates into 2 subunits that can reassemble on another mRNA molecule to restart protein synthesis

Describe the Ubiquitin Proteasome System

• Ubiquitin:

  • small protein (peptide)

• Proteasome:

  • Large cylindrical structure that degraded proteins via proteases that cleave peptide bonds

• Process:

  • Polyubiquitination:

    • Ubiquitin (small protein) that is continuously added to protein to target it for proteasome

  • Proteasome:

    • Little tube w/ lid on either end

    • the ubiquitin chain targets the protein to the lid

    • the protein fed through tube ( filled with hydrolytic enzymes) to break a part peptide bonds

    • Results in peptide fragments that can be reused