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biochemistry 2


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Makes 2 cuts on either side of DNA & utilizes helicase & polymerase I to remove pyrimidine dimers
Uses UV light to cleave pyrimidine dimers (THF & FADH2 cofactors)
DNA photolyase
tags oldest DNA strand
DAM Methylase
Removes uracil produced by deamination of cytosine
Uracil-DNA glycosidase
mechanism turned on by autolysis of LexA repressor
SOS response
Increased mutation % as a result of increased protein production
SOS response
Catalyzes pairing of ssDNA w/ complimentary dsDNA
coprotease for LexA
List correct order of recBCD generation of ssDNA for recombination:

A)hits chi site & releases D
B)enters at free ends
C)BC unwinds DNA using 2 ATP/base pair
D)ssDNA coated w/ DNA-binding protein
Stabilizes ssDNA during replication
SSB protein
DNA repair system utilizing Dam methylase, MutH, L, & S proteins, DNA helicase II, SSB, DNA polymerase III, Exonuclease I, & DNA ligase
Mismatch repair
Repair system that corrects abnormal bases, alkylated bases, & pyrimidine dimers
Base-excision repair
DNA repair mechanism that utilizes DNA glycosylases, AP endonucleases, DNA polymerase I, & DNA ligase
Base-excision repair
DNA repair mechanism that utilizes ABC excinuclease, DNA polymerase I, & DNA ligase
Nucleotide-excision repair
DNA repair mechanism that corrects DNA lesions that cause large structural changes (pyrimidine dimers)
Nucleotide-excision repair
DNA repair mechanism that utilizes DNA photolyases & O6-methyllguanine-DNA methyltransferase
Direct repair
DNA repair mechanism that corrects pyrimidine dimers, O6-methylguanine
direct repair
DNA repair mechanism that corrects errors left by polymerase III
Mismatch repair
8-oxoguanine DNA glycosylase-1 correlates strongly (x5-10) w/ what kind of risk?
cancer (both smokers & nonsmokers)
Why does DNA have Thymine instead of Uracil?
It would be impossible to distinguish uracil from deaminated cytosine
DNA repair mechanism lacking in xeroderma pigmentosa patients
nucleotide excision repair
condition with skin lesions, cancer, photosensitivity (allergy to light)
xeroderma pigmentosa
DNA absorbs light at a wavelength of:
260 nm
activated when RNA polymerase stops at a deformity, which recruits excision repair enzymes
Transcription Coupled Repair (TCR)
gene for susceptibility to breast and ovarian cancer
responsible for accumulation of mutations causing a large proportion of breast and ovarian cancers
BRCA1-altered TCR
inhibits recombination when mismatches are high, inducing apoptosis
type of recombination that occurs mostly during meiosis (eukaryotes) and conjugation (prokaryotes)
homologous recombination
type of recombination that doesn't change the order of genes but determines which alleles will be linked.
List in order the steps of the Holliday model
1-homologous DNA are alligned
2-breaking of strands for crossing-over
3-heteroduplex DNA extended by branch migration
4-two strands of intermediate are cleaved, gaps are filled
catalyzes strand assimilation reaction (forms d-loop) at the expense of ATP
hydrolyzes ATP and unwinds dsDNA for ssDNA production
nuclease that recognizes a chi site
resolvase that cuts the holliday intermediate in one or the other plane
produced by the process of recombination
multimers & concatemers
type of recombination used by bacteriophage λ integration
site-specific recombination
Diversity of genes depends on communication between. . .
Serve to inactivate antibiotics, metabolize natural products, and produce toxins
Encodes conjugation function, found in male bacteria
F-factor plasmid
Resistant Transfer Factors (RTF)
R-factor plasmids
upon integration they duplicate recipient DNA (not inverted)
Transposons (jumping genes)
coded by transposons and makes staggered cuts in DNA
A double-stranded nucleic acid that exhibits semiconservative replication has heavy nitrogen at all positions. After heavy nitrogen source is taken away, the strand is replicated through 3 generations. what is the composition of the strands?
7 strands with no N14, 1 strand with half N14 (12.5%)
Enzyme that unwinds the DNA strands at the growing replication fork
DNA helicase
Relaxes tension in DNA by breaking one strand, unwinding it, and then ligating it. Alters DNA by a single linking number
Topoisomerase I
Relaxes tension in DNA by breaking both strands, unwinding them, and then ligating them. Changes the linking number in steps of 2
Topoisomerase II
Generates negative supercoils (requires ATP) in E.coli as well as relaxes the tension associated with helicase activity
DNA gyrase
Prevent DNA strands from reforming their base pairs after helicase passes until the polymerase synthesizes the new strand.
SSB proteins
Measure of level of supercoiling in DNA=

Linking number of completely relaxed =
=Linking number

103 kDa, 3'-5' exonuclease, 5'-3' exonuclease, processes 200 nucleotides, functions in replication & repair
DNA polymerase I
88 kDa, 3'-5' exonuclease, processes 10000 nucleotides at a rate of 10 bases/sec, functions in DNA repair
DNA polymerase II
900 kDa, 3'-5' exonuclease, processes 500000+ nucleotides at a rate of 1000 bases/sec, functions in DNA replication
DNA Polymerase III
Located in eukaryotic nucleus, has exonuclease, functions in replication, primase complex
DNA polymerase α
Specific change in DNA nucleotide sequence
Located in eukaryotic nucleus, doesn't have exonuclease, functions in replication and repair
DNA Polymerase β
Located in eukaryotic mitochondria, has exonuclease, functions in replication and repair
DNA Polymerase γ
Located in eukaryotic nucleus, has exonuclease, functions in replication and repair
DNA Polymerase δ
Generates the primer in the initiation complex, resulting in an RNA-DNA hybrid
Enzyme that joins 3' OH to a 5' phosphate to restore integrity of DNA strands
Strand that appears to move 3' to 5', but actually moves 5'-3' creating Okasaki fragments
Lagging Strand
DNA replicates in what phase of the cell cycle
Synthesis phase
Primers needed for leading strand=

Primers needed for the lagging strand=

E.coli replication constituent=SSB

Eukaryotic replication constituent=
E.coli replication constituent=primase

Eukaryotic replication constituent=
primase/polymerase α
E.coli enzyme that removes primer=Pol I/RNase II

Eukaryotic enzyme that removes primer=
Process in which mutations manifest
Organism altered by mutation
Single mutation at a single base site (actually two after replication)
Point mutation
mutation that results in an amino acid change; a change in the codon triplet that is unmasked by degeneracy
Mutation that changes an amino acid codon triplet into a stop codon
Mutation in which one or more nucleotides are deleted
Mutation in which one or more nucleotides are inserted
Point or missense mutation that is silent except under certain conditions
conditional mutation
mutation that permits the phenotype of the mutant to revert back to the wild-type
One purine is changed to the other or one pyrimidine is changed to the other
A purine is substituted for a pyrimidine or vice versa
Mutation seen in sickle cell anemia
point mutation (Glutamate switched to
Mutation seen in Marfan's syndrome
Point mutations in the fibrillin gene (FBN1)
autosomal dominant disorder affecting fibrous connective tissue (FBN1 gene)
Marfan's syndrome
Change in the register in which amino acid sequence is read
mutations that occur in non-gamete cells (not inherited)
mutations that occur in gametes (inheritable)
test for detection and classification of mutagens and carcinogens
Ames test
In an Ames test, S. typhimurium supplemented with histidine is cultured. Plates A and B are given a possible carcinogen. Plate C is a control. Plate A shows growth of colonies. Is the substance carcinogenic?
First predictor of longevity=

Second predictor of longevity=
=DNA repair capacity

=Immunological capacity
uvrABC removes pyrimidine dimers by making two cuts; ___ nucleotides from the 5' end and ___ nucleotides from the 3' end

=Removal of pyrimidine dimers

=defective DNA displacement

=patch (two enzymes)


=Polymerase I & Ligase
Enzyme that uses UV light to cleave pyrimidine dimers (uses THF & FADH2 cofactors)
enzyme that methylates the oldest DNA strand
Dam methylase
consequence of the action of Dam methylase
old strand recognized by Mut H & S; new strand is repaired
Enzymes that recognize methylated strands of DNA
Mut H & S
Nucleoside produced by cytosine deamination
Enzyme that removes uracil produced by deamination of cytosine via glycosidic bond-cleavage
Uracil-DNA glycosidase
Process by which a cell deems it more necessary to stay alive than to prevent mutation
SOS response
Enzyme that represses SOS response
Recogize 4,6,or 8 base-pair palindromes
Restriction Enzymes
Sequence with bilateral symmetry (read the same 3'-5' as 5'-3')
Source of restriction enzymes
Limits foreign DNA integration into cells, made from restriction endonuclease & Dam methylase
Restriction enzymes
Copy sections of DNA, then inserts desired gene
Monitors extent of DNA damage in humans and induces apoptosis (inhibits recombination) if there is too much mismatch
Technique for separating DNA fragments by size
In electrophoresis, phosphate groups move toward what?
anode (+)
State of plasmid that replicates independently of the chromosome
State of plasmid that replicates only when the chromosome does
_____ plasmids produce continuously (high copy number)
Vectors that provide larger fragments (~20kb) and more efficient delivery to the host
viral vectors
Vectors (~1mb) that contain Autonomously Replicating Sequences (ARS), Centromeres, Telomeres, Selectable markers, and a MCS
Yeast Artificial Chromosomes (YACs)
Chromosome digested with restriction enzymes & ligated with a plasmid, so each vector will have only one piece. The genome is represented by many pieces each in a different cell
Genomic Library
Enzyme that mRNA converted to dsDNA in order to assemble a cDNA library
Reverse Transcriptase
Formed when products of Reverse Transcriptase are cloned into vectors, displaying only expressed sequences of genes
cDNA library
Central dogma of molecular biology
DNA ->(Trasncription)->RNA->(Translation)->Protein
=nondiscriminatory (copies everything)

=selective transcription (certain genome parts)
DNA replication=

RNA replication=
Two functions of DNA in prokaryotes=
=structural information (genes)

=regulatory signals (promoters, terminators, etc.)
Requirements for RNA polymerases
-DNA template
-4 ribonucleotide triphosphates
-divalent cations (Mg++)
Three required steps for RNA synthesis
step in which RNAP finds a promoter & unwinds DNA (most regulated step)
=site of eukaryotic transcription
=site of eukaryotic translation
Primary sigma factor for most genes (housekeeping)
Sigma factor involved in transcribing nitrogen-regulated genes
Sigma factor responsible for heat shock genes
stationary phase Sigma factor
Sigma factor that regulates expression of flagellar genes
Unique polymerase in that it can initiate de novo synthesis (no primer required, but it needs a promoter)
Makes only ribosomal RNA and actually synthesizes a long preribosomal rRNA transcript that is a precursor for the 18S, 5.8S, and 28S rRNAs (Eukaryotic)
does most of the work associated with transcription; makes all mRNA (Eukaryotic)
makes all tRNAs and the 5S rRNA (Eukaryotic)
Regulatory element located outside the promoter that decrease higher-order DNA structure (chromatin)
Transcription factor that can be cut out & spliced in somewhere else without functional loss
Step that most transcription factors operate
Serve to increase higher order DNA structure (chromatin)
___________ acetylate histones (loosen chromatin)

___________ de-acetylate histones (tighten chromatin)

tightly controlled genes in E.coli to be "on" only when lactose is present
lac operon
states of lac operon:

=glucose present, lactose absent

=glucose present, lactose present

=glucose absent, lactose present
lac operon "off"=

lac operon "on"=

lac operon "highly on"=
set of genes in tandem that are regulated by a single promoter
Site bound by repressor to prevent transcription
small molecule that relieves repression allowing RNAP access to the promoter
required to turn lac operon "highly on" by activation
CAP (catabolite activator protein) or
CRP (cAMP receptor protein)
HIV-1 promoter
protein RNAP I binds to in Termination
termination that involves only an interaction between RNA and RNAP
rho independent (intrinsic)
Pause site hit by hairpin loop that signals termination is rich in what base pair?
forms a hexamer that binds to RNA at a hairpin pause structure; moves along RNA and unwinds RNA-DNA duplex to terminate transcription
contains a 5' cap structure and a 3' poly A tail
consists of methylated guanine triphosphate attached to the OH group on the ribose at the 5' end of mRNA
200 adenine nucleotides attached to 3' OH end of mRNA
poly A tail
associate with proteins to form ribosomes
16S, 23S, and 5S rRNA is derived from __________, while 18S, 28S, 5S, and 5.8S rRNA is derived from ___________.

cloverleaf strux, contains modified nucleotides including thymine
The primary tRNA transcript is shortened at both ends via
_____ cleaves 5' end of tRNA

_____ cleaves 3' end of tRNA

(both ribozymes)
=RNase P

=RNase D
sequence at 3' end of tRNA that binds the amino acid
adds CCA to 3' end of tRNA after RNaseD cleavage
terminal deoxytransferase (nucleotidyl transferase)
___________ have operons

___________ have introns and exons

type of RNA processing that differs between prokaryotes & eukaryotes
prevents mRNA degradation & allows translation; linked by 5'-5' triphosphate
5' cap
enzymes required for 5' cap addition:
1- phosphohydrolase (P cleavage)
2- guanylyltransferase (5'-5'
phosphate attack using GTP)
3- guanine-7 methyltransferase
(protects 5' end)
4- 2'-O-methyltransferase (unique to
process by which introns are removed and exons are linked to encode a functional polypeptide
Self-splicing introns=

Spliced via lariat structure=
=Groups 1 & 2

=Group 3
RNA-protein complexes contain ______ made by RNAP III that enable lariat structure formation
small nuclear RNAs (snRNAs or Snurps)
The lariat structure is created when the _____ end of ribose attacks the phosphodiester bond at the ______ forming a covalent link

Finally, the ______ nucleophilically cleaves the intron.
=2' OH

=5' cleavage site

=3' OH (on severed 5' exon)
allows for diversity of structure and function without duplication of genes, depending on environmental need
alternative splicing
function of the poly A tail=
protection and translatability
three steps of polyadenylation=
1-precursor RNA cleavage via
polyadenylation signal
2-poly A tail addition via poly A
3-unneeded 3' end material is degraded
genetic code:
____ bases
____ possible codons
____ reading frame
____ reading direction
=3 bases/amino acid
=5'-3', N-C
Start codon=
=AUG (codes for methionine)
define degeneracy.
the genetic code has more than one codon for each amino acid, differing in only the third base
Stop codons=
Steps of translation=
=Activation, Initiation, Elongation, Termination
=synthesis of aminoacyl-tRNAs
=assembly of mRNA and initiator aminoacyl-tRNA on the ribosome
=Synthesis of peptide bonds in RNA translation
=Release of newly synthesized peptide bonds and disassembly of translation apparatus
set by the start codon (AUG)
reading frame
allows one tRNA to translate more than one codon
wobble pairing:
U=A or G
G=C or U
I=A, C, or U
E.coli ribosomes consist of __ and __.

Eukaryotic ribosomal subunits are ___ and ____.
E.coli=50S and 30S (70S)

Eukaryotes=60S and 40S (80S)
measure of how rapidly ribosomal subunit migrates in an ultracentrifuge (dimensions of size & shape)
Svedburg units
Site of protein synthesis
Activated intermediate for protein synthesis
control gene expression by blocking mRNA
RNAmi or RNAi
Unravels and seeks out mRNA to cleave at the attachment site
RNAmi or RNAi
Main problem of RNAi in treatment of disease
site of activation reaction
cytosol (not on ribosome)
tRNAs which have different sequences but carry the same amino acid
isoaccepting tRNAs
only tRNA recognized by eIF-2
When a methionine residue is to be located anywhere in a protein other than the N-terminus, it is brought to the ribosome by:
Euk. Prok.
1st AA=
Euk. Prok.
1st AA=Met Formyl Met
IF=12 3
Ribosomes=80S 70S
Aminoacyl-tRNAs are brought to the ribosome as a _________with GTP and the elongation factor eEF-1
ternary complex
Incoming aminoacyl-tRNA base pairs its anticodon with mRNA codon located in the ___ site of the ribosome
Elongation steps=
1-binding (A site)
2-translocation (A->P; mRNA movement)
catalyzes formation of peptide bond
peptidyl transferase
occurs when stop codon moves into the A site
recognizes stop codons and triggers the hydrolysis of newly sythesized protein from tRNA at the P site
Release factor
complex that translates mRNA by several ribosomes at the same time
binds the small ribosomal subunit and causes incorrect anticodon-codon pairing
blocks the A site
inhibits peptidyl transferase (also affects mitochondria)
causes premature termination of translation
inhibits EF-2 (translocation) via ADP-ribosylation
Diptheria toxin
activated under conditions of low heme levels in order to prevent protein synthesis
heme-regulated inhibitor (HRI)
Kinase phosphorylates (with ATP) the ___ inhibiting protein synthesis
eIF-2-GDP complex

(inhibits formation of eIF-2-GTP)
mutation in exon 11 of the gene for the α-chain of β-hexoaminidase A
Part of tRNA that reacts w/ mRNA
anticodon loop
Energy Requirements for AA synthesis:

__ Activation
__ Initiation
__ Elongation
__ Termination
1 ATP/AA for Activation
1 ATP(E) & 1 GTP(P) for Initiation
[(# of AA-1)*2] GTP for Elongation
1 GTP for Termination
for 100 AA, 200 GTP, 100 ATP (P), 101 ATP(E)
The difference between SER & RER
RER has ribosomes
Oligosaccharides added to the amide N of an Asp residue
N-linked oligosaccharides
oligosaccharide attached to the O of a Ser or Thr residue
O-linked oligosaccharide
What type of oligosaccharide are mucus proteins?
What type of oligosaccharides are Blood serum proteins?
The core of all N-linked oligosaccharides consists of--
2 glcNAc and 3 Man residues
A core oligosaccharide is derived from what lipid-linked oligosaccharide intermediate?
dolichol phosphate
The 14-hexose precursor of N-linked oligosaccharides is assembled on the dolichol phosphate starting with the transfer of glcNAc-1-phosphate from --
UDP-glcNAc (cytosolic side of ER membrane)
After the target sequence has been translocated into the lumen of the ER, the N-linked oligosaccharide precursor is transferred where?
Asn residue part of the sequence Asn-X-Ser or Asn-X-Thr
Dolichol-P + 2 UDP-GlcNAc------>UMP + UDP + Dolichol-PP-2GlcNAc is inhibited by what antibiotic? (first step of N-linked synthesis)
Translocation of N-linked oligosaccharide intermediates occurs at which step?
signal that the protein is ready for transfer to the Golgi
removal of 3 glc and 1 man while glycoprotein is still in the ER
GalNAc transfer to a Ser or Thr residue from UDP-GalNAc takes place in what type of synthesis?
O-linked oligosaccharide
Most secreted proteins and membrane proteins are of what type?
What sugar do glycoproteins never have?
Targeting sequence for the ER membrane
Targeting sequence to send proteins to mitochondria
N-terminal sequence
Targeting sequence to send proteins to nucleus
Basic amino acid rich
Targeting sequence to send proteins to lysosome
Targeting sequence to send proteins to peroxisome
Carboxyl terminal SerLysPhe
The process by which proteins enter cells from the blood
Protein that coats transferin-filled vesicles
reacts with a specific receptor and becomes incorporated into endosomes that deliver iron to ferritin
protein that stores iron in the cell
determines the half-life of cytosolic proteins
amino-terminal residues
binds with protein, then binds with lysine residues to form an isopeptide linkage for degradation
contain tryptic and chromotryptic-like proteases for degradation
type of linkage of salivary mucin
Sialic acid (NANA) attaches to what part of oligosaccharides?
dictates the structure of glycoproteins
target of transferase in protein glycosylation
nucleated sugar
site of completion of modifications to N-linked oligosaccharides
Linkage of blood group glycoproteins to cells
O-linked to Ser
Terminal linkage of H blood group antigen
Fucose (alpha 1,2)

Terminal linkage of A blood group antigen
GalNAc, Fuc (alpha 1,3)
Terminal linkage of B blood group antigen
Gal, Fuc (alpha 1, 3)
Reacts with antibodies:
Phenotype: O
Genotype: O/O
Reacts with antibodies: none
Antibodies: anti-A, anti-B
Phenotype: O
Reacts with antibodies:
Phenotype: O
Genotype: O/O
Reacts with antibodies: none
Antibodies: anti-A, anti-B
Phenotype: A
Reacts with antibodies:
Phenotype: A
Genotype: A/A, A/O
Reacts with antibodies: ANTI-A
Antibodies: anti-B
Phenotype: B
Reacts with antibodies:
Phenotype: B
Genotype: B/B, B/O
Reacts with antibodies: ANTI-B
Antibodies: anti-A
Phenotype: AB
Reacts with antibodies:
Phenotype: AB
Genotype: A/B
Reacts with antibodies: ANTI-A, ANTI-B
Antibodies: NONE (universal acceptor)
signal for golgi to send glycoproteins to vesicles
enzyme defect in I-cell disease
GlcNAc transferase
GlcNAc transferase
enzyme defect in I-cell disease

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