bio 303 exam 3

C Value paradox

the complexity of an organism is not related to its genome size (amount of DNA it contains or its “C Value”

genome size can range greatly in some groups of organism

supercoiled DNA

DNA twisted beyond the typical double helix shape

does this to save space, relieve stress/pressure from the tight winding of DNA, and allows DNA to be opened up

relaxed DNA circle

typical DNA structure with 10 base pairs per complete turn

underwound DNA circle

contain a bubble (or bubbles) on unpaired bases that compensate for underwinding

negatively supercoiled DNA- easier to open up DNA strand for replication, meiosis, mitosis, etc

Topoisomerase II

enzyme that untangles a pair of DNA molecules by cleaving one DNA duplex and passing the other duplex through the gap

upper jaw grabs one duplex (not cleaved) and the lower jaw grabs the second duplex (which it cleaves) and passes the first through the split second DNA molecule

bacterial chromosomes are organized into…

supercoiled loops

each loop can be independently relaxed or condensed

four proteins (HU- wrapping, FIS and IHF- bending, HNS- compaction) involved in supercoiling into a higher order structure

EX: each loop of the DNA duplex is twisted around itself in a right handed direction- results in negative supercoiling

• supercoiling can only be relaxed when the loop is nicked (and only the nicked loop will be affected)

eukaryotic DNA is organized into nucleosomes

each nucleosome consists of two of each H2A, H2B, H3, and H4 histone proteins, a segment of DNA containing about 200 nt, and one molecule of H1

molecule of H1 is a linker- outside of 8 histone molecule complex and connects DNA wrapped around the histone proteins

histone proteins

small proteins (100-200 AA)

five major types: H1, H2A, H2B, H3, H4

20-30% of AA are lysine + arginine- positively charged (**DNA neg charged)

both DNA-histone and histone-histone binding are important for chromatin structure (bc they make up nucleosome structure)

sequence of histones are conserved during evolution

nucleosomes compact into…

chromatin fibers- spring-like/coiled structure

bead line structure of nucleosomes only seen in artificial conditions, typically it is compact into chromosome fibers in the nucleus

stages of DNA condensation

DNA duplex —> nucleosomes (histone proteins wrapped with DNA) —> chromatin fibers (DNA form during interphase) —> chromosomes (DNA form for meiosis/mitosis- higher order)

chromosome territories

less dense chromosome domains/contain fewer genes- outskirts of the nucleus or close to the nucleolus (very center of the nucleus)

more dense chromosome domains/contain more genes- further inside the nucleus (but between the nucleolus and the outskirts of the nucleus)

polytene chromosomes

highly repeated DNA sequences that never separate

produce large amounts of RNA and proteins quickly

nucleoid

bacteria cells (no nucleus + one circular chromosome as DNA)

DNA forms a multiply looped structure- supercoiled and compact

eukaryotic DNA consists of three major components

30-75%: unique sequences

• one copy of each unique sequence

5-45%: highly repetitive sequences

• as many as 10^5 copies per genome

1-30%: middle repetitive sequences

• 10-1000 copies per genome

some parts of DNA condense more/less than others because they are different sections of different complexity

repetitive nucleic sequences in eukaryotic genomes

some components have a base composition that is different from the average

satellite DNA: components with unusually low/high G+C contents, fairly short nucleotide sequences that may be tandemly repeated up to a million times

• repeats are separated from other DNA because their density is different

renature more readily than nonrepetitive DNA because they consist of high similar or identical sequences

heterochromatin

highly repetitive and low complexity

condense faster because of the high amount of repeats

located on the middle of chromosomes near centromere and on the ends where telomeres are (orange section)

molecular structure of the centromere

specific region of the eukaaryotic chromosome that becomes visible as a narrow constriction along the condensed chromosome

holocentric chromosome

very large

have centromeric sequences spread throughout their length

also called a diffuse centromere

localized centromere

conventional type

microtubules attach to a single region on the chromosome- kinetochore

point centromeres: found in different yeasts, more repetitive

regional centromeres: found in other eukaryotes, may contain hundreds of kilobases or repetitive DNA

point centromere

yeast centromere

centromere DNA has four centromere determining elements (CDEs)

CDEs: CDE1, CDE2 (more variable than 1 and 3), CDE3 (~25 base pairs), CDE4 (variable sequences) = ~220 base pairs total (centromere)

spindle fibers connect t kinetichore proteins

telomere

special DNA protein structure located at each end of a linear chromosome

consists of tandem repeats of simple sequences and associated rpoteins

essential for chromosome stability

this region cannot be synthesized in DNA replication

telomerase

RNA molecule which serves as the template for telomere DNA synthesis

adds telomeres to the end of the chromosome

mechanism to match age of cells (stops DNA from becoming too short over time as replication occurs and the telomere is lost)

complexity of a DNA sequence

the number of possible pairing partners for any DNA strand present in a given quantity of DNA

compare complexity of 2 strands using same amount of DNA

repetitive sequence = less complex than a random sequence

shorter sequence = less complex than a longer sequence

denaturation

heating of DNA leads to denaturation of the two strands

rate depends of the G+C content (3 Hydrogen bonds are harder to break than the 2 between A+T)

repetitive DNA sequences renature…

more rapidly than unique sequences (also denature faster)

easier to find pairing partners/arrange back into the proper sequence when repeated/less complex

rate of DNA renaturation depends on..

concentration and sequence complexity

low complexity = faster

high concentration = faster

short DNA strand = faster

rates of renaturation of DNAs with different kinetic complexities

longer sequences = more DNA = longer to renature

C0t curves for eukarytoic genomes typically have three distinct steps

highly repetitive, middle repetitive, and unique sequences

number of copies of a sequence is inversely related to the obersved (uncorrected) C0t1/2 values for each class

fundamental concepts for semiconservative DNA replication

replication of DNA by strand separation

each parental strand remains intact

each parental strand serves as a template for the synthesis of a complementary strand

copying of the 2 template strands using A-T and G-C base pairing

end result of DNA replication is the formation of two daughter duplexes that are identical to the parental duplex

experimental proof of semi conservative replication of DNA

density of daughter DNA duplexes less than that of the parental strand- becomes “lighter” (15N of parental DNA —> 14N of daughter DNA)

after one round of DNA duplication: the molecules contain one template/parental strand and one new/daughter strand, so they have an intermediate density due to the combination of the red 15N and blue 14N strands (red and blue DNA molecules)

after two rounds of DNA duplication: the molecules containing only the 14N appear lighter (all blue DNA molecules)

proved DNA replication is semi conservative

Meselson and Stahl

DNA becomes less dense over time due to semiconservative replication

use of a thymidine analog (BRdU) provides cytological proof that DNA replicates in a semi conservative manner

BRdU detects sister chromatid exchange during DNA replication

one round of replication: each chromatid has one old strand (normal DNA) and one new strand (contains BRdU)

two rounds of replication: one chromatid is fully contains BRdU and other has one strand of each

BRdU can visually detect recombination events:

• uniform staining = no exchange

• patchy staining = sister chromatid exchange occurred

homologs can undergo crossing over

recombination between sister chromatids of homologous pairs

three different replication models

theta replication: e. coli

rolling circle replication: DNA phages

multiple origins and bidirectional replication: eukaryotic chromosomes

theta DNA replication

both parental DNA strands remain intact

DNA replication begins at specific sequences: “origins of replication”

replication fork: where the DNA is unwinding to allow new strand to form complimentary to template strand

can be

• unidirectional: moves in one direction

• bidirectional: moves both directions simultaneously

rolling circle DNA replication

one of the template DNA strands is cut by enzyme nuclease to create a primer 3- OH end

nucleotides are added to the 3’ end of the cut strand (bases added in 5’ —> 3’ direction)

for every round of replication, the tail of the rolling circle becomes one unit longer

okazaki fragments

small chunks of the new DNA strand created as the lagging strand because of the direction DNA must build (onto the 3’ end on new strand)

because lagging strand builds away from replication bubble, must build in pieces as replication fork advances

replication of a linear eukaryotic chromosome

replication begins at replication origin and is bidirectional

multiple replication bubbles can exist and they will fuse once they meet

critical steps in DNA replication

unwinding, stabilization, and stress release

initiation my a Primosome Complex

chain elongation and proofreading

DNA replication proteins

helicase: enzyme, unwinding DNA double helix

SSB proteins: stabilizing the templates (single stranded DNA), DNA stressed when unwound and no longer double stranded

gyrase or topoisomerase: release mechanical stress of unwinding

primosome: initiate new strand synthesis

DNA polymerase complex: elongating new strand

DNA ligase: ligating (combining) Okazaki fragments

e.coli DNA replication fork

helicase unwinds DNA at replication fork

RNA primer initiates new strand synthesis

SSB proteins stablizie the single stranded DNA

DNA polymerase adds the complimentary nucleotides

DNA ligase joins fragments on lagging strand

gyrase cleaves DNA ahead of replication fork to release stress

prevention of knotting of DNA by DNA gyrase

double strand broken and the helix can swivel to release stress before replication bubble reaches this section of DNA

priming of DNA synthesis with a RNA segment

primer needed for DNA polymers (complimentary DNA bases to the template strand) to have a section of nucleotides to attach to to begins replication

new DNA chains are initiated by short RNA primers- RNA primer becomes 5’ end of new strand and is complimentary to the 3’ end of the template strand

DNA polymerase can attach the new DNA nucleotides to the RNA primer that begins the synthesis of the new strand

addition of a deoxynucleotide (DNA nucleotide) to the 3’ OH end

replication occurs in the 5’ —> 3’ direction (new strand), bases are added to the 3’ end of the template strand

leading strand vs lagging strand

leading strand: moves towards the replication fork and is synthesized continuously in one large fragment

• 3’ strand of the template strand- DNA can just be added to the new strand easily

lagging strand: moves away from the replication fork and is synthesized in many, smaller Okazaki fragments (which are glued together by ligase)

• must add to 3’ of template strand, which is the side of the strand the replication fork is moving towards

joining of Okazaki fragments

removal of RNA primer

replacement of the primer with the correct DNA sequence

joining where the adjacent DNA fragments come into contact

DNA polymerase in DNA replication

can add free nucleotides only to the 3’ end of the newly forming strand (so strand is built in the 5’-3’ direction)

can add a nucleotide only to a preexisting 3- OH group

correcrs mistakes in newly synthesized DNA (proofreading)

addition of a dideoxynucleotide to the 3’ OH end of a DNA chain…

terminates chain elongation

dideoxyribose: no 3’ OH group, so DNA replication cannot continue

Sanger Sequencing

takes advantage of ddNTP chain terminators (N = some nucleotide, A, T, C, G)

DNA replication will be stopped where these are added- where whichever nucleotide the ddNTP is located

proofreading exonuclease function of DNA polymerase

when an incorrect base pair is formed, an enzyme cuts the DNA so that the incorrect nucleotide will be removed- ex: A-G removed because complimentary pairing should be A-T

endonucleases: enzymes that cleave the phosphodiester bond within a polynucleotide chain

exonucleases: enzymes that cleave phosphodiester bonds at the end of a polynucleotide chain

• DNA polymerase can act as a 3’-to-5’ exonuclease

central dogma of biology

DNA —> RNA —> Protein

(transcription) (translation)

three major types of proteins

enzyme proteins- speeds up/facilitates reactions

regulatory proteins- regulate processes

structural proteins- make frame of cells

proteins

composed of one or more polypeptide chains

each polypeptide chain is a series of covalently joined amino acids

20 different amino acids commonly found in polypeptides, can be joined in any order and in any number (typically 100-1000)

structure of an amino acid

Amino group (H2N), R group (various), Carboxyl group (COOH), and Hydrogen all attached to a central carbon (alpha carbon)

carbon backbone formed from central carbons

structure of amino acids depend on their R groups

some amino acids positively charged, some are negatively charged based on their R groups

• positive: have extra amino group in R group

  • Alanine, Lysine

• negative: have extra OH group in R group

  • Aspartic Acid, Glutamic Acid

• charged: polarized molecules

  • hydrophilic (have charge) vs hydrophobic (have no charge)

amino acids are joined by _____ bonds to create polypeptides

peptide bonds

R groups jut off the carbon backbone

amino acids are added to the carboxyl (-COOH)end of the polypeptide

dehydration synthesis reaction

water removed when two amino acids combined in order to form a peptide bond —> forms dipeptide chain

as more amino acids added —> formed polypeptide chain

sickle cell anemia

normal red blood cells are smooth and round, move easily through blood vessels to carry oxygen to all parts of the body

sickle shaped cells are stiff and sticky, do not move easily through the body and tend to form clumps/get stuck in blood vessels

DNA and protein are colinear

DNA sequence determines amino acid sequence in a point-to-point manner

mutation in DNA can be traced back to a change in the AA in the DNA

basis for genetic disease

transcription

process by which the information contained in DNA is copied into a single strand RNA molecule of complementary base sequence

RNA polymerases

large, multiple subunit complexes who active form is called the RNA polymerase holoenzyme

bacteria: one RNA polymerase transcribes all genes

eukaryotes:

• RNA polymerase I- rRNA

• RNA polymerase II- mRNA and certain small RNAs

• RNA polymerase II- tRNA, 5S rRNA

RNA synthesis

DNA serves as template

U base pairs with A in RNA synthesis (rather than T)- complementary base pairing with template strand

still builds onto the 3’ end of the growing new strand

RNA polymerase does not require a primer- can initiate instantly

• can recognize unique region of DNA to begin RNA synthesis: promoter sequence

e. coli promoters have conserved sequences

-35 and -10 nt regions determine promoter strength

consensus sequences

share sequence homology, but not identical

sigma factor protein: help to recruit RNA polymerase to promoter region

the more closely a sequence resembles these sequences, the stronger the promoter (???)

eukaryotic RNA polymerase II promoters contain a…

TATA box- much further from from transcription start (25-30 nucleotides) than in e. coli

farther so there is more control

stages of transcription

1. chain initiation: RNA polymerase binds to initiate transcription

2. chain elongation: only the template strand is transcribed, transcription bubble is ~15 nt long with 8-9 nt paired with the 3’ end of the RNA

   1. nucleotides added to the RNA strand are complimentary to the template strand (but code for the non template strand)

3. termination: RNA polymerase reaches a terminator sequence and the RNA and polymerase are released

basal factors required to form the class II pre-initiation complex

6 factors and RNA polymerase II = preinitiation complex

factors:

• TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

• many are multi subunit

• factors and RNAP must also bind in a specific order (in vitro)

transcription chain initiation by RNA polymerase II

transcription chain initiation by RNA polymerase II

TBP- TATA box binding protein

TBP (TATA box binding protein) binds to and distorts DNA using a…

a beta sheet inserted into the minor groove- bends DNA ~90 degrees and forces open the minor groove

TBP-DNA complex

the TBP is in purple, the DNA TATA sequence is in gray

functions of TFIIA, TFFIIB, TFIID, and TFIIF, TFIIE, TFIIH

TFIIA: binds to TBP and could be considered a TAFII (TBP associated factor)

TFIIB: needed for the polymerase/TYIIF complex to bind to TFIID- linker between the two

TFIIF: binds to the RNAP and reduces non specific binding of the RNAP to DNA

TFIIE: binds after polymerase/TFIIF binds and stimulates and recruits TFIIH

TFIIH: required for promoter clearance, has DNA helicase activity for melting DNA at transcription bubble, has Kinase activity for phosphorylation of the CTD of the large subunit of RNAP

promoter clearance

chain termination

1. self termination: most common in bacteria, transcription stops when the polymerase encounters a particular sequence of nucleotides

2. termination requires the presence of a termination protein

self termination sequences contain…

a hairpin and U-rich region

some terminators also require a terminator protein

initiation of a second rounds of transcription…

does not need to wait for the completion of the first

how many DNA strands are transcribed for any one gene?

only one

though, different genes can be transcribed on different strands

in bacteria, transcription and translation are…

coupled

structure of messenger RNA

RNA processing is aided by RNA polymerase itself

RNA processing

the conversion of a primary transcript into an mRNA or tRNA molecule

includes splicing, cleavage, modification of termini, and (in tRNA) modification of internal bases

prokaryotes: primary transcript is mRNA

eukaryotes: primary transcript must be processed

steps of RNA processing

5’ guanine cap- addition of a 7-methylguanosine to the 5’ end of the RNA strand

3’ polyadenylation- addition of ~200 nucleotide polyA tail (stops enzymes from destroying strand in cytoplasm)

splicing- removal of intron and joining of exons

5’ capping functions

1. regulation of nuclear export

2. prevention of degradation by exonucleases

3. promotion of translation (attachment to ribosome)

4. promotion of 5’ proximal intron excision

snRNPs

small nucleoprotein particles that mediate splicing

RNA splicing takes place in nuclear particles known as spicesomes

snRNPs are composed of proteins and several specialized small nuclear RNA (snRNA) molecules

mechanism of RNA splicing

introns have 5’ GU and 3’ AG splice sites and an internal branch site A

splice site mutations may result in…

the retention of an intron

ribozyme (RNA enzyme or catalytic RNA)

RNA molecules possessing a well defined tertiary structure that enables it to catalyze the chemical reaction in RNA splicing

many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds or the hydrolysis of bonds in other RNAs

3’ polyadenylation functions

1. nuclear export

2. translation

3. stability of mRNA

mRNA

provides the nucleotide coding sequence that determines the amino acid sequence

ribosomes

responsible for protein synthesis

tRNA

adaptor molecules that decode the nucleotide sequence to amino acid sequence

aminoacyl tRNA synthetase

attaches an AA to tRNA (based on complimentary anti codon to the codon on mRNA)

initiation, elongation, and release factors

specialized proteins that aid in each stage of translation

mRNA is translated into protein

structure features of tRNA

1. the 5’-terminal monophosphate group

2. the acceptor stem is a 7-base pair stem made by the base pairing of the 5’-terminal nucleotide with the 3’-terminal nucleotide

3. the CCA tail is important for the recognition of tRNA by enzymes critical in translation

4. the anticodon arm is a 5-bp stem whose loop contains the anticodon

5. the D and T arm secondary structures are important for the function of tRNA

translation initiation in prokaryotes

initiation of protein synthesis in eukaryotes

A Site: the ribosomal site which binds the incoming aminoacyl-tRNA (acceptor site)

P Site: the site which holds the peptide tRNA, that is the tRNA which is covalently linked to the growing polypeptide chain (Peptidyl site)

E Site: a site which transiently binds to the outgoing, deacylated tRNA (exit site)

elongation is a repeated cycle of three processes

1. bringing each aminoacylated tRNA into line

2. forming the new peptide bond to elongate the polypeptide

3. moving the ribosome to the next codon along the mRNA

translation termination occurs when…

a stop codon is reached

transcription and translation are ______ in bacteria

coupled

in eukaryotes, transcription takes place in the nucleus and translation occurs in the cytoplasm

prokaryotic ribosomes can initiate at ____________ in a mRNA

internal sites

prokaryotic mRNAs- can be polycistronic

• ribosomes can initiate translation within an mRNA, therefore making multiple polypeptides

eukaryotic mRNAs- are only monocistronic

• ribosomes must initiate translation at the 5’ end, therefore only one polypeptide can be made at a time

translation in prokaryotes vs eukaryotes

transcription and translation

• prokaryotes: simultaneous and colocalized

• eukaryotes: separate (nucleus and cytoplasm)

initiation

• prokaryotes: f-met

• eukaryotes: Met

ribosome binding

• prokaryotes: SD dequence

• eukaryotes: 7 me-G Cap

polycistronic mRNA

• prokaryotes: common

• eukaryotes: rare or nonexistent

RNA processing

• prokaryotes: no

• eukaryotes: yes (5’ G Capping, 3’ PolyA tail, splicing)

primary protein structure

sequence of residues making up the protein (chain of amino acids)

secondary protein structure

refers to local sub-structures: alpha helix and beta strand or beta sheets (initial folding of AA chain)