BIOC EXAM 1

Can the same enzyme bind to both 3' and 5'?

No

Bond formed by bases

H bond

Bond formed by sugar and phosphate

Covalent ( causes polarity)

DNA double helix features

- antiparallel
- complementary bases in each strand
- 1 helix turn \=10bp
-major/minor grooves

Draw sugar phosphate backbone

Configuration of nitrogenous bases

Planar

Where are the nitrogenous bases?

In the middle(creates favorable stacking energy bc planar)

Alpha helix vs DNA double helix

AA r groups stick out and bases point inward

Hydrogen Bond

Weak electrostatic interaction bc of polarity of covalently bound H and 2 EN atoms

What happens in hydrogen bonding?

H is cov bound to en atom and forms bond w another en
H takes part + charge and en atoms part -

Strongest H bonds

Straight O—H|||||||O

H bond donors

OH or NH

H bond acceptors

O or N

Major/minor groove features

-glycosidic bonds don't directly oppose (asymmetry)
-major groove can fit proteins

How do proteins find specific dsDNA sequences?

- major groove
-Can distinguish cg vs gc and at vs ta
-determine bases through hbond donor/acceptor and methyl patterns

How do proteins access the bases to ID sequences?

DNA binding proteins ( read NA info) (TFs)
- alpha helix of HTH domain fits in major groove
- these proteins touch major and minor grooves

Multivalency

Build up of AA side chain interactions by DNA binding proteins
(20+ weak contacts become strong)

Bonds of DNA binding proteins in the major groove

H bonding
Ex: asp to A arg to G

Most common DNA form

B form

Other DNA forms

A form (wider and more compact)
Z form ( left handed)
3strand(hoogsten bp)

RNA vs DNA

DNA:
info storage
Double stranded
Antiparallel
AT
Deoxyribose
Chargaff

RNA:
info storage,info transport, catalysis
Single or double or triple
Parallel or anti
AU
No chargaff
Ribose

What model describes the secondary structure of tRNA?

Cloverleaf

What base pairing does cloverleaf do?

Watson Crick
( can have open circles as long as base pairing maintained)

How is the tertiary structure of tRNA formed?

More H bonding (long range interactions)
Non watson crick

How to clone a DNA fragment

Insert into vector and propagate in E. coli

Enzymes used to manipulate DNA

Nucleases
DNA ligase
DNA polymerase
Polynucleotide kinase
Phosphotase

What do nucleases do?

cut DNA

Endonuclease

Cleave dna internally by cutting phosphodiester bb

Exonuclease

Cuts DNA molecule on either end

DNA ligase

Glue
Join DNA molecule end to end by catalzkng phosphodiester bond between 3' OH and 5' PO4

DNA polymerase

Synth DNA from template strands

Polynucleotide kinase

add triphosphate to 5' OH

Phosphatase

Removes 5' triphosphate

Restriction enzyme

Endonucleases in prokaryotes that fight over invading viruses recog and cleaving dna seqs

Modification enzyme

Modifies host dna through methylation (restriction enzymes with cut unmethylated dna)

How to restriction enzymes recognize their sites?

*Bind to DNA and scan for recog seq (bind will change the DNA conform)
*multiple backbone contacts with major and minor grooves
*AAs h bond to phosphates in bb and bases
They cut at specific sequences which are usually 4, 6, or 8 base palindromes
Frequency of restriction sites is 4^n (n\=bases)

Popular digestion enzyme

HindIII

How do dna frags reanneal?

Bc of sticky ends ( overhanging bases)
Ligase will glue the breaks with phosphodiester bond in bb

What are the necessary features of a vector?

- origin of replication (sequence that allows replication in e coli cell)
-selectable marker ( gene to tell which bacteria have the vector)
- can accom additional DNA ( big and small frags)
- unique restriction site where foreign DNA is inserted

How does gel electrophoresis separate DNA frags?

Fragments migrate according to size (constant mass charge ratio)
Diff agarose concentrations allow diff band resolutions

Which gels for large and small frags?

Large: agarose 100nt-10kb
Small: acrylamide 10-50nt

How to create a human DNA library?

1) isolate DNA from lymphocyte and part digest with HindIII and digest vector with HIII
2) mix vector + insert and DNA ligase
3) transform DNA into e coli and select for recomb DNA (on agar with selection compound and galctosidase substrate

Use double selection

Or just use PCR

cDNA library vs genomic library

Cdna: cloned rev transcribed mRNA ( no introns expressed genome ) - if genomes or related is known can map to referemce
Genomic: large DNA frags ( whole genome) - want to do if genome seq is unknown

How to design primers to amp dna seq?

What must a DNA strand have to grow?

3' OH group (primers have this)

How are dideoxynucleotides used in dna seq?

Sanger sequencing
Replication stops w/o 3' OH
*We use them to block further DNA molecule growth
Add to reaction mix has H instead of OH on 3' and prevent phosphodiester bonding

Draw dieoxynucleotide

DNA synth direction

5 to 3
Only add new subunits to 3'

How can you use short seq to build a complete seq?

Short overlapping seqs come together as contigs
Software will overlap snd configure

How big is the human genome?

3x10^9 base pairs ( billion)

C-value paradox

lack of correlation between genome size and the biological complexity of an organism (we don't know why)

Hyperchromisity

Single stranded dna absorbs higher uv than double (cot)

Cot procedure

1 shear dna into fragments
2 denature at 95 c
3 reassociate through slow cooling at different concentrations (Co) and rates of time (t1/2)
4 measure percent reassociated to ds

Cot anaylsis

Simple dnas/more repeated sequences reassociate faster
-larger genomes are slower
Human DNA is different because different fragments reassociate as different times
Unique seqs long time

ORF qualifications

Over 100 AAs
Start atg

E. Coli genome features

Circular dna
1 origin if rep
Wall to wall genes
No introns
Operons
Few repeats
Horizontal gene transfer recent

How do you find genes in a primary sequencing read?

Prok:easy bc no introns, count in between and stop at the right stop, look for recognizable promoter
Euk: hard, scan with computer find start and try to count 100 triplets before stop codon

Yeast genome features

Wall-wall genes
16 chrom, mito, and multiple origins
No operon
Some repeated genes
A few introns
More gene density than humans

How many genes in the human genome?

20,000 (splice to make more proteins)

What percent of our genome is related to gene control and what percent encodes for proteins?

20 percent & 1-1.5

How many open reading frames in the human genome?

About 20,000

Simple repeats

Repeated seqs 1-30 nuc found between or inside genes

Types of simple repeats

Mononucleotide
Dinucleotide: highly polymorphic ( good genetic markers )
Trinucleotide: disease causing
VNTRs: good at iding individuals

Which simple repeat causes diease?

Trinucleotide
REPEATS EXPAND WITH EACH GENERATION( need to relax dna repair fidelity)
CCG or CAG in coding or non coding dna are linked to disease like huntingtons (more is higher risk)

DNA only transposons: stucture frequency movement

Cut and paste genes from one area of the genome to another until they become inactive (fossilized)

LINES: structure frequency movement

20% of genome
Most abundant class of repeated DNA
1-9kb
L1 most common 6.5 kb (only 50 functional rest only transpose)
Non retroviral transpons
L1s move when transcribed by rna poly II to make mrna which is translated into nuclease and reverse transcriptase copies it back to dna

Which repeated DNA encodes reverse transcriptase and endonuclease?

LINES

SINEs

13% of genome
10^6 Alu element is most common (restriction site for RE Alu1)
- 300 nuc, poly A tail, GC rich
Use same reverse transcriptase as LINEs
Transcribed by rna pol II but don't encode, just make small tRNA
Alu element looks like 7s rna

Why can SINES cause diease?

.01% disease
The alu elements can be insertional mutagens when they jump
Our enzyme will methylate the C in the CpG gene which decreases transcription ( turns off genes )

Third most abundant class of repeated DNA

Retroviral like elements

Types of satellite DNA

Centromere and telomere
\# repeats can change from uneven crossing over

Repeated genes and how they arose

mRNA, tRNA, histones
Duplicated during evolution bc cell needs many copies
Globin, growth hormone, and albumin common ancestors
1 gene duplicates and both may slight change

Pseudogenes

former genes that have accumulated mutations and are nonfunctional

How do genes duplicate???

Unequal crossing over, slippage

Reverse transcriptase mechanism

Copies RNA into DNA
Host uses no such enzyme so good inhibitor target (HIV)
adds nuc onto 3' end of of poly chain, but chain must have 3' OH to add more nuc
5' triphosphate (dntp) is used and attacked first

Design chain terminating inhibitor

Cant be like dntp bc of side fx
Make a dideoxy!!!
Resemble substrate

DNA replication basics

1 each strand copy just once
2 time and location or initiation and replication very carefully controlled
3 rep must begin at an origin euk many prok 1
4 bidirectional with rep fork
5 2 strands rep differently
6 DNA pols need primer at least 20 nuc
7 many proteins help
8 low error rate proofreading by dna pol

Draw nucleotide linkage condensation mechansm between 3'oh and 5' po4

Why is there a leading and lagging strand?

DNA polymerase can only add nucleotides in one direction (5' to 3')
No adding to 5' end

What proteins are needed for initiation?

1 initation protein
2 helicase
3 ssb
4 primase
5 clamp loading complex
6 sliding clamp
7 DNA pol III (prok only)

Why proteins are need for elongation?

1 DNA pol III
2 DNA pol I
3 DNA ligase
4 topisomerase

Why does helicase need atp?

Has to unwind dna by breaking a lot of h bonds
(Hexamer)

SSB

Binds to strands to prevent reanealing and unwanted base pairing, only contacting bb

Clamp loader

Couples with atp to load sliding clamp on dna

Sliding clamp

Complex that circles and holds strand so dna pol doesn't fall off

Primase

(Rna pol) Makes the rna primers for dna pol to bind to ( primers about 10 nu long)
Doesn't need a primer and can start anywhere

Ligase

Seals the nicks in the dna frags
Hydrolyzes atp and activates nuc on 5' side of nick

Which enzymes work more on the lagging strand?

Primase & ligase dna pol I? Clamp loader all

How many primes does the leading strand need?

1

What enzyme recognizes primers and replaces them with dna?

DNA pol I (prok) or pol alpha (euk)

What is supercoiling and how is it fixed?

When you unwind the dna it gets supercoiled on the outside
Supercoiling is resolved by topoisomerases
- cuts one strand of dna to be rotated freely around the other then resealed (cov bind to phos w/ tyrosine)
Use en of super coil

Fill out replication fork cartoon

Replication in eukaryotes is similar except for what?

Couple to cell cycle

How many mistakes does dna pol make and why so few?

1 per billion bp
They have 3' to 5' exonuclease activity
Backs up and repairs in opp direction of polymerase removing mismatches (back up and chew)
Proofreading constitutes second active site

How does a cell know when to start replication?

Cell cycle regulation/origin licensing

How do cells duplicate w/o introducing error?

High fidelity polymerases, proof reading, and post replication repair systems

How does mismatch repair know which strand is new?

Old is methylated
All the new strand is removed and resynth

Telomerase

Extends the lagging strand to compensate shortened chrom ends

Why is complete replication of the lagging strand difficult?

Cant replicate chrom ends bc of 5' degradation, we would need an rna primer
Ends get recognized as ds breaks and get shorter w/ each cell division (dna pol chew out with no upstream dna to extend)

How does telomerase extend the lagging strand?

Has its own template that binds to the ss end overhang of parent dna, adding repeats
Dna pol and primase then synth to original length