BIOL 308 Midterm 1: 2-11

What are the 3 main functions of DNA?

Store information

Replicate faithfully

Ability to mutate

gene

entire DNA sequence necessary to produce functional protein or RNA

Whats the main concept of the central dogma of biology?

Replication of DNA

transcription of RNA

Translation proteins

Which step in the central dogma can proceed in reverse?

transcription

What types of information does DNA carry?

Coding and regulatory sequences

what is the purpose of coding sequences

for proteins and RNA

what is the purpose of regulatory sequences

signals or binding sites

what is the initiation of gene expression

transfer of information

what is the template strand also known as?

antisense strand, the one which gets transcribed

what is the sense strand ressemble?

identical to RNA

DNA strands have….?

polarity, is complimentary and antiparallel

Replication of DNA is ____

semiconservative

how did Meselson and stahl determine semiconservative replication

grew e.coli for many generations in N15 medium then transferred to N14 medium, DNA was placed in centrifuged and density revealed the presence of heavy DNA, hybrid DNA, and light DNA

2 distinct bands of DNA formed as generations went on

As meselson and stahl continued their experiment, what did they notice in future generations?

less and less hybrid DNA was present, more light DNA appeared

What is the outcome of semiconservative replication?

one strand serves as template to synthesize new daughter strand, information is preserved

how do mutations prove the concept of colinearity of genes and prtoeins

mutations alters the coding sequences of proteins = change in amino acid

what is the importance of mutations?

formation of new alleles, variability in genes

name the 2 purines

Adenine and guanine

name the 3 pyrimidines

cytosine, thymine, uracil

What is the structural difference between DNA and RNA

DNA lacks an oxygen on the sugar, only 1 OH group

What makes the structure of RNA unstable?

the presence of OH on the 2’ ribose sugar, single stranded

Where is the most energy stored in a nucleotide

in the phosphate groups, more phosphate carries more energy

How are phosphates attached to the sugars in DNA chains?

phosphodiester linkages

how are DNA sequences read?

5’-3’

Chargaff’s Rule

#purines = #pyrimidines

structure of B DNA

has major and minor grooves

completes 1 turn in 3.4 angstrom

Why are there major and minor grooves

binding sites of regulatory factors

nucleotide sequences reveal unique areas of acceptors and donors

what forces help form DNA double helix

rigid phosphate backbone

van der waals interactions

hydrophobic interactions between backbone and base

ionic interactions, salts stabilize phosphate backbone

hydrogen bonding between base pairs

how does triple helix occur?

when purines make up one strand, pyrimidines the other

a third strand is accommodated

how many base pairs does it take for one turn of B DNA

10 bp

is B DNA right or left handed?

Right

why is chromosomal DNA a dynamic structure

can have flipped bases for a short segment

allows for recognition of DNA in gene expression and DNA repair

if DNA is denatured, when does renaturation occur?

if DNA sequences are complementary, non complementary strands will NOT hybridize

What are factors that denature DNA

Heat

low ionic strength promoting repulsion of backbones

High pH NaOH strips H+ shared between bases

competitive functional groups outcompeting H-bonding between bases

covalent modifications preventing H-bonding

enhancing solubility of hydrophobic substance

What are 2 ways to monitor DNA denaturation

by viscosity or absorbance

how does absorbance tell us about DNA denaturation

there is lower absorbance in dsDNA than ssDNA

What is Tm?

temperature where 50% of DNA is denatured

How does the curve of absorption and DNA denaturation resemble and why?

sigmoidal curve, eventually plateaus as all DNA is denatured to ssDNA

what is the hypochromic effect

stacked bases in dsDNA have lower absorbance

what is the hyperchromic effect

unstacked bases causes increase in absorbance

What is Tm a function of?

GC content, more GC = higher Tm needed

why does GC increase Tm?

more energy to break apart 3 H-bonds compared to AT (2 H-bonds)

Why does higher salt = higher Tm?

Salt binds to negatively charged phosphate backbone, DNA becomes neutral and strands dont repel each other so they are harder to separate

What is renaturation/hybridization?

recombination of 2 complementary ssDNA

What is renaturation dependant on?

DNA concentration

salt concentration for repulsion forces

temperature, cold is better

time

size of DNA/length

complexity of sequences

What does Cot analysis measure?

the complexity of a genome and amount of repetitive DNA in a sample

What does it mean if a genome has no repeating sequences?

it is unique

how are units of complexity measured?

by #of unique nucleotides

What are the steps of performing Cot Analysis?

Shear control DNA and unknown DNA

Denature both using heat

Allow both to cool and re-anneal

measure absorbance of ds & ss DNA

plot curve

Why do we use Cot instead of Time on X-axis?

We will see a measure of concentration not complexity

Which type of sequences will anneal the fastest? Which will anneal the slowest?

highly repetitive, unique

Describe the relationship of reassociation and genome size

inversely proportional, larger genomes take longer (if unique)

Is the complexity of the genome correlated with the biological complexity of the organism?

No, some prokaryotes have larger genomes than humans

Where are circular genomes commonly found?

prokaryotes, chloroplasts, mitcochondia

How does circular DNA denature?

nicking occurs during DNA replication, can also be introduced experimentally by an enzyme

Which common features do circular and linear DNA share?

1º structure= sugar phosphate chain

2º structure = double helical structure

3º structure = supercoil complex with proteins

What is a benefit of supercoiled DNA?

reduces stress on DNA by twisting/untwisting double helix

What are topological isomers?

DNA differing in states of supercoiling

When does DNA become linearized?

in replication and transcription

When does supercoiling occur if both strands of DNA are fixed?

tension will be relased by forming supercoils every 10bp

What is positive supercoiling?

dsDNA opened in front of opening, forming tight positive left hand supercoils

What is negative supercoiling?

dsDNA opened behind opening, loosens and untwists causing negative right handed supercoil

How to determine twisting #

T = total # of base pairs/ # of base pairs per tern (10)

What does twisting # measure

how tightly the helix is woundW

What is writhing #?

number of superhelical turns, how many times DNA crosses over itself

Negative supercoil = -

positive supercoil = +

What is Linking #?

L = T+W

how many times one strand of closed dsDNA encircules the other strand

What is the function of topioisomerases?

enyzmes that recognize and regulate supercoiling to help replication and transcription

What is the supercoiling of most cell DNAs

negatively supercoiled, they store energy by untwisting double helix

what happens when DNA is overwound

positive supercoiling, reduced DNA protein interactionwh

What happens when DNA is underwound?

negative supercoils, store energy for strand separation

What does topiosomerase I in prokaryotes do?

relaxes negative supercoiling by nicking one strand, changes L# in steps of 1

What does topioisomerase II in prokaryotes do?

relaxes positive supercoiling by cutting both strands, changes L# in steps of 2

What does gyrase do in prokaryotes?

introduces negative supercoils

Whaat does reverse gyrase do?

stabilizes genome, protects DNA strand

What is mRNA?

messenger RNA, specifies order of amino acids for protein synthesis

What is tRNA?

transfer RNA, during translation mRNA is interpreted by tRNA

WHat is rRNA

ribosomal RNA, helps in translation

What are small RNAs?

have regulatory functions

What are ribozymes?

RNA with enzymatic functions (splicing, peptide bond)

Why are there different conformations of RNA?

carry variety of functions out in the cell

What type of helix is RNA?

A Helix

What prevents formation of B Helix in RNA?

2’ OH group

What is the secondary structure in RNA?

fold in on themselves to forms hairpins

GU will pair for extra self-complementation

Why are bases modified in RNA?

to facilitate alternate base pairing

What is tertiary structure in RNA?

L shape formed by interactions of secondary structure

high rotational freedom in non-pairing regions

What is unconventional about tertiary structure in RNA

formation of triple base pairing from free H present

What type of knots can form in tert. structure of RNA?

pseudoknots where base pairing sequences dont line up

What are additional conformations in Tert. RNA?

A-minor motif

tetraloop motif

ribose zipper motif

kink-turn motif

kissing hairpin loop

What is the average diameter of a bacterial cell?

1 um

Whats the problem with E.coli cell structure and the amount of DNA it has?

The DNA is 1.36nm long and cannot fit within the cell if chromatin is not altered

How is the bacterial chromosome structured?

compact genome structure called the nucleoid

What is the nucleoid?

A supercoiled, circular molecule of DNA with polyamines, HU proteins(binding)

Where can supercoiling occur in bacteria?

in space or around proteins

How do Eukaryotes pack 2.5m of DNA into a ball 10um in diameter?

eukaryotic chromatin wraps tightly around a histone protein to form the unit: nucelosome

Whats the chromosome organization called?

beads on a string

beads = histone in nucleosome core

Where are histones found in eukaryotes?

in ALL eukaryotic nuclei

What is the structure of histones?

small lysine and arginine (basic) rich proteins with 5 major subunits

Describe the organization of histone subunits

2 dimers form 2 tetramers, 2 tetramers form an OCTOMER

What is a nucleosome?

octet of histones wrapped in DNA

How can size of DNA wrapped around octet be analyzed?

using DNase by partial digestion