BIOCHEM EXAM 2

phosphodiester bond

joins nucleotides together 3’ to 5’

Why are major and minor grooves important?

allow enzymes to H bond for replication

semi-conservative

one parent strand conserved with each daughter strand

In what direction is DNA synthesis in replication?

5’ to 3’

What do DNA polymerases require in replication?

template strand, primer with free 3’ OH group, 4 dNTPs

What direction is DNA exonuclease proofreading activity?

3’ to 5’

processivity

number of nucleotides added before polymerase dissociates

What direction is DNA pol I nuclease activity?

5’ to 3’

nick translation

removes primers with 5’ to 3’ exonuclease activity and fills gaps with DNA with 5’ to 3’ polymerase

Which subunit of DNA pol III is responsible for high processivity?

beta

DnaB

helicase; unwinds DNA

ssB

single-stranded DNA binding protein; binds ssDNA to stabilize and protect it

primase

makes RNA primers

Which polymerase does most of the DNA synthesis

DNA pol III

alpha subunit of DNA pol III

5’ to 3’ polymerase that adds DNA

epsilon subunit of DNA pol III

3’ to 5’ exonuclease proofreading activity

beta subunit of DNA pol III

clamp for high processivity

Function of DNA pol I

nick translation

DNA ligase

seals nicks

Ori C

DNA replication initiation site

For initiation of DNA replication, ___ binds to __ , causing __. Next, __ loads __

DnaA (AAA+ATPase) binds OriC, opening DNA unwinding element (DUE); DnaC (AAA+ATPase) loads DnaB (helicase)

Termination of replication at ___ sequence with __ protein

Ter sequence; Tus protein

Steps of Cytosine Deamination Repair (Base Exclusion Repair)

1) Uracil DNA glycosylase removes uracil base

2) AP endonuclease makes nick

3) DNA pol I does nick translation

4) DNA ligase seals nicks

What is needed for Sanger dideoxy chain termination:

ssDNA template, radioactive primer, 4 dNTPs, 4 test tubes, DNA polymerase

Steps of Sanger Sequencing Method:

* termination of synthesis creates fragments


1. run oligonucleotides on polyacrylamide gel
2. autoradiography (put gel on xray film)
3. read sequence 5’ to 3’ from bottom

Big Base Teqchnique

adaptation of Sanger method suing fluorescent dyes instead of radioactivity

What is needed for Illumina Sequencing:

template DNA on flow cell, universal primer, reversible terminator nucleotides, 9 degree N DNA pol

Illumina Sequencing steps:

1) flow reversible terminator nucleotides; 9 degree N adds one

2) flash and record color

3) chemically remove fluorophore and blocking group

4) repeat

What makes up a reversible terminator nucleotides?

fluorophore and 3’ blocking group

When to use Sanger vs Illumina sequencing techiniques:

Sanger - high fidelity, sequence one gene or plasmid

Illumina - faster mass sequencing and high throughput, entire genome

mRNA

messenger; working copy of DNA gene; template for protein synthesis

tRNA

transfer; translates mRNA codons into amino acid sequence of protein

rRNA

ribosomal; major component of ribosome; 23s rRNA IN E.coli catalyzes peptide bond formation

snRNA

small nuclear; carries out splicing in eukaryotes

siRNA

small interfering; cleaved from dsDNA by DICER; binds to mRNA as part of RISC and degrades mRNA

What does RNA polymerase need for prokaryotic transcription?

template and 4 NTPs

What direction is transcription synthesis?

5’ to 3’

template strand vs non-template (coding) strand

template - used to make mRNA

coding - same sequence as mRNA with T instead of U

What subunit of RNA polymerase does the promoter bind?

sigma

what is the start site of prokaryotic transcription?

promoter

What are the promoter elements of prokaryotic transcription?

10 region (Pribnow Box), 35 region, UP element

What is the Pribnow box of promoter bound by?

sigma 70 of RNA pol

What is the UP element of promoter bound by?

alpha subunit of RNA pol

consensus sequence

shows nucleotides that are most common in each position

What makes a strong promoter?

if promoter is identical to consensus sequence

How does rho-independant termination of transcription in prokaryotes occur?

hairpin and oligo U

How does rho-dependant termination of transcription in prokaryotes occur?

1) rho (ATP dependent helicase) binds rut (rho utilization sequence)

2) rho moves up RNA and pulls it away from polymerase

What polymerase is used in eukaryotic transcription?

RNA polymerase II

What are the eukaryotic promoters?

TATA box and Inr (initiator)

What is required for RNA pol II to bind to promoter?

transcription factors TBP or TFIIs

5’ cap

7-methyl guanosine; added during transcription to enhance stability and translation

3’ polyA tail

cleavage of mRNA downstream of specific signal and polyadenylation to enhance stability and translation

polyadenylation signal function

cleavage of new RNA and 3’ polyadenylation

splicing

removal of introns from genes

What ribozyme catalyzes the splicing of introns?

snRNA

What forms splicesomes?

snRNPs (snRNA + protein)

ribozyme

RNA enzyme

alternative splicing

more than one 5’ or 3’ splice site

poly A site choice

more than one poly A tail

reverse transcriptase

enzymes that HIV can use to make dsDNA from an RNA template

telomerase

enzyme that contains RNA and synthesizes DNA from the RNA to make chromosome ends

start codon

AUG

stop codons

UAA, UAG, UGA

wobble hypothesis

third base of codon forms less stringent pairs

prokaryotic ribosome subunit composition

50s large subunit + 30s small subunit = 70s ribosome

What are ribosomes made of?

rRNA and protein

which rRNA subunit aligns ribosome with shine delgarno sequence

16s

which rRNA subunit in prokaryotes is the peptidyltransferase (forms peptide bonds)

23s

aminoacyl tRNA synthetase

attaches amino acids to tRNA

Use _____ hydrolysis to charge tRNAs with amino acid

ATP

What is the first amino acid for bacteria?

formylmethionine

What is the first amino acid for eukaryotes?

methionine

What is the direction of synthesis along protein and mRNA in translation?

N to C; 5’ to 3’

start signal for prokaryotic translation

shine delgarno sequence

What are the components of prokaryotic translation initiation

IF1 and IF3 bind 30s subunit to bind mRNA; IF2-GTP binds to recruit fmet-tRNAfmet; 50s associates and IF2 hydrolyzes GTP to dissociate

EF-__ delivers an aminoacyl tRNA to ribosome

EF-Tu

EF-_ recycles EF-_

EF-Ts recycles EF-Tu

Translocation of ribosome is carried out by ___ using energy from __ hydrolysis

EF-G’ GTP hydrolysis

__ hydrolyzes to release peptide in translation

peptidyl-tRNA link

What allows ribosome to dissociate in termination of translation

EF-G, ribosomal recycling factor (RRF), GTP hydrolysis

What subunits make up the eukaryotic ribosome?

60s large subunit + 40s small subunit = 80s ribosome

Which rRNA subunit is peptidyltransferase in eukaryotes?

28s

How does eukaryotic translation occur?

mRNA is held in a circle by PAB and eIFs

How is eukaryotic translation initiated?

ribosome binds at 5’ cap and begins at first AUG

general method of cloning

1. cut vector and target DNA with restriction endonuclease
2. combine in presence of DNA ligase
3. transform host cell
4. propagate

Type II Palindromic restriction endonuclease

recognize palindromic sequences on DNA and makes staggered cut “sticky ends”

multiple cloning site (polylinker)

piece of DNA with multiple restriction sites

vectors

accept foreign DNA and replicate in host cell

how do plasmids transform cells

put plasmid and bacteria in O degree celsius CaCl2 then jump to 37 degrees celsius

What is needed for plasmid to replicate in host?

OriC, antibiotic resistance gene, and restriction enzymes

expression vectors

make foreign protein in E.coli

What is required for expression vectors?

OriC, promoter, ribosome binding site (RBS) shine delgarno sequence, multiple cloning site, transcription stop signal (rut), antibiotic resistance

cDNA library

eukaryotic genes made from tissue mRNAs using reverse transcriptase and DNA pol I for expression vectors

What must be used for expression vectors using eukaryotic genes in E.coli? Why?

cDNA because eukaryotic genes have introns

Golden Gate Cloning

DNA fragments have been domesticated…


1. Type IIS restriction enzyme cuts outside of its recognition sequence
2. specifically designed overhangs allow annealing of consecutive DNA components
3. T4 ligase joins pieces

DNA domestication

1) use PCR primers to change the sequence

2) remove internal restriction site

type IIs restriction endonuclease

used for golden gate cloning; recognize a site of 4-8 nucleotides; cleave at a defined site that is different from the recognition site

what is PCR

polymerase chain reaction; amplifies DNA without cloning in host cell

What polymerase is used for PCR

heat-stable Taq polymerase

PCR steps

1. heat to denature
2. cool to anneal primers
3. heat to polymerization step using Taq