BIOC EXAM 1

studied byStudied by 0 People
0.0(0)
Get a hint
hint

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

1/158

Tags & Description

Studying Progress

New cards
158
Still learning
0
Almost done
0
Mastered
0
158 Terms
New cards

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

No

New cards
New cards

Bond formed by bases

H bond

New cards
New cards

Bond formed by sugar and phosphate

Covalent ( causes polarity)

New cards
New cards

DNA double helix features

  • antiparallel

  • complementary bases in each strand

  • 1 helix turn =10bp -major/minor grooves

New cards
New cards

Draw sugar phosphate backbone

New cards
New cards

Configuration of nitrogenous bases

Planar

New cards
New cards

Where are the nitrogenous bases?

In the middle(creates favorable stacking energy bc planar)

New cards
New cards

Alpha helix vs DNA double helix

AA r groups stick out and bases point inward

New cards
New cards

Hydrogen Bond

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

New cards
New cards

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 -

New cards
New cards

Strongest H bonds

Straight O—H|||||||O

New cards
New cards

H bond donors

OH or NH

New cards
New cards

H bond acceptors

O or N

New cards
New cards

Major/minor groove features

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

New cards
New cards

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

New cards
New cards

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

New cards
New cards

Multivalency

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

New cards
New cards

Bonds of DNA binding proteins in the major groove

H bonding Ex: asp to A arg to G

New cards
New cards

Most common DNA form

B form

New cards
New cards

Other DNA forms

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

New cards
New cards

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

New cards
New cards

What model describes the secondary structure of tRNA?

Cloverleaf

New cards
New cards

What base pairing does cloverleaf do?

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

New cards
New cards

How is the tertiary structure of tRNA formed?

More H bonding (long range interactions) Non watson crick

New cards
New cards

How to clone a DNA fragment

Insert into vector and propagate in E. coli

New cards
New cards

Enzymes used to manipulate DNA

Nucleases DNA ligase DNA polymerase Polynucleotide kinase Phosphotase

New cards
New cards

What do nucleases do?

cut DNA

New cards
New cards

Endonuclease

Cleave dna internally by cutting phosphodiester bb

New cards
New cards

Exonuclease

Cuts DNA molecule on either end

New cards
New cards

DNA ligase

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

New cards
New cards

DNA polymerase

Synth DNA from template strands

New cards
New cards

Polynucleotide kinase

add triphosphate to 5' OH

New cards
New cards

Phosphatase

Removes 5' triphosphate

New cards
New cards

Restriction enzyme

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

New cards
New cards

Modification enzyme

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

New cards
New cards

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)

New cards
New cards

Popular digestion enzyme

HindIII

New cards
New cards

How do dna frags reanneal?

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

New cards
New cards

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

New cards
New cards

How does gel electrophoresis separate DNA frags?

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

New cards
New cards

Which gels for large and small frags?

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

New cards
New cards

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

New cards
New cards

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

New cards
New cards

How to design primers to amp dna seq?

New cards
New cards

What must a DNA strand have to grow?

3' OH group (primers have this)

New cards
New cards

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

New cards
New cards

Draw dieoxynucleotide

New cards
New cards

DNA synth direction

5 to 3 Only add new subunits to 3'

New cards
New cards

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

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

New cards
New cards

How big is the human genome?

3x10^9 base pairs ( billion)

New cards
New cards

C-value paradox

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

New cards
New cards

Hyperchromisity

Single stranded dna absorbs higher uv than double (cot)

New cards
New cards

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

New cards
New cards

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

New cards
New cards

ORF qualifications

Over 100 AAs Start atg

New cards
New cards

E. Coli genome features

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

New cards
New cards

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

New cards
New cards

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

New cards
New cards

How many genes in the human genome?

20,000 (splice to make more proteins)

New cards
New cards

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

20 percent & 1-1.5

New cards
New cards

How many open reading frames in the human genome?

About 20,000

New cards
New cards

Simple repeats

Repeated seqs 1-30 nuc found between or inside genes

New cards
New cards

Types of simple repeats

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

New cards
New cards

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)

New cards
New cards

DNA only transposons: stucture frequency movement

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

New cards
New cards

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

New cards
New cards

Which repeated DNA encodes reverse transcriptase and endonuclease?

LINES

New cards
New cards

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

New cards
New cards

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 )

New cards
New cards

Third most abundant class of repeated DNA

Retroviral like elements

New cards
New cards

Types of satellite DNA

Centromere and telomere # repeats can change from uneven crossing over

New cards
New cards

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

New cards
New cards

Pseudogenes

former genes that have accumulated mutations and are nonfunctional

New cards
New cards

How do genes duplicate???

Unequal crossing over, slippage

New cards
New cards

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

New cards
New cards

Design chain terminating inhibitor

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

New cards
New cards

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

New cards
New cards

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

New cards
New cards

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

New cards
New cards

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)

New cards
New cards

Why proteins are need for elongation?

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

New cards
New cards

Why does helicase need atp?

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

New cards
New cards

SSB

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

New cards
New cards

Clamp loader

Couples with atp to load sliding clamp on dna

New cards