Shared Flashcard Set


bch 401G test 4
bch 401G test 4
Undergraduate 3

Additional Biochemistry Flashcards




-__ is the process in which the DNA within a cell makes an exact copy of itself.
-- __: cell cycle arrested
-- __: cellular contents, excluding the chromosomes are duplicated
-- __: Each of the 46 chromosomes is duplicated by the cell
-- __: the cell double checks the duplicated chromosomes for error, making any needed repairs
- dna replication
-- G0
-- G1
-- S
-- G2
dna replication is __
- dna replication in ecoli requires more than __
- five of these are __, which catalyze the synthesis of new dna
- __ and __ play key roles in dna replication
- 20 proteins
- dna polymerases
- poly 3; poly 1
- polymerase I: __ removal and dna __; __ and __ exonuclease
- polymerase II: function is __, __ exonuclease, attachment of __ to __
- polymerase III: fuction is __, __ exonuclease
- polymerase IV: function is __; attachement of __ to __
- polymerase V: function is __, attachment of __ to __, __, and __
-primer; repair; 5>3; 3>5
- repair; 3>5; bulky hydrocarbons; bases
- replication; 3>5
- repair; bulky hydrocarbons; bases
- repair; bulky hydro carbons; bases; sites missing bases; covalently joined bases
polymerases __, __, and __ can replicate through regions of damaged DNA. they are called __ polymerases or error-prone polymerases
2;4;5; translesion
__ catalyzes __ bond formation
dna polymerase; phosphodiester
important characteristics of dna synthesis
1. __ and __ are required
-- these include __, __, __, __
2. a __ is used to direct dna synthesis
-- __ is a __ enzyme
3. a __ from which the new strand grows must be present
1. deoxynucleoside triphosphates; mg+2
-- dATP; dGTP; dCTP; dTTP
2. template strand
-- dna polymerase; template directed
3. primer
chain elongation is a __ reaction
- base pair between incoming __ and a residue of the __
- terminal __ attacks __ of incoming nucleotide to form a new __ linkage
- nucleotides added one at a time to the __ end
- chain grown is always __
nucleotidyl group transfer
- deoxynucleotide 5' triphosphate; parental strand
- 3' OH; alpha phosphorous; phosphodiester
- 3'
- 5>3
important characteristics of dna synthesis
4. many __ have nuclease activity that allows for the removal of mismatched bases
- __ activity contributes to the remarkably high fidelity of dna replication, less than 10^-8 errors per base pair
- most uncorrected replication errors are subsequently corrected by the __
4. dna polymerases
- 3>5 exonuclease
- dna repair enzymes
proofreading by the __ activity of __ during dna replication
3>5 exonuclease; dna polymerases
dna polymerase structure
- the structures of dna polymerases are __
- the region containing the active site approximates the shape of a __
- the __ and __ domains wrap around the dna positioning it in the active site located in the __ domain
- similar
- right hand
- thumb; fingers; palm
-Specificity of Replication is Dictated by the __ of __
-The specificity of replication is determined not only by the correct __ between the incoming dNTP and the DNA template, but also by the overall __ of the incoming base.
- an analog of adenosine that cannot form h bonds still directs incorporation of __
- complementarity; bases
- hydrogen bonding; shape
- t
__ Contributes to Fidelity
-Binding of the incoming __ induces a structural change in which the __ domain closes over the nucleotide, forming a pocket into which only the correct nucleotide fits.
induced fit
- dNTP; finger
-__, powered by __, separate the strands of the double helix to make the DNA available for __.

-The __, which consists of a ring-like structure composed of __ subunits, acts as a wedge to pry the helix apart.
-helicases; atp hydrolysis; dna polymerase
- helicase; six
Defects in the helicase can result in __, a pathological condition characterized by premature aging.
Werner syndrome
__ Prepare the Double Helix for Unwinding
-When DNA is unwound for replication, -__ occurs because of overwinding in nearby regions of the helix.

-__ induce or eliminate supercoils.

-__ relax supercoils while __, such as DNA gyrase in E. coli, introduce supercoiling.
- supercoiling
- topoisomerases
- type I topoisomerases; type 2 topoisomerases
__ is a Therapeutic Target
-__ and __ are inhibitors of DNA gyrase that are used to treat bacterial infections.
-__ prevents anthrax poisoning
bacterial topoisomerase
- nalidixic acid; ciprofloxacin
- ciprofloxacin (cipro)
dna replication is highly __ and very __
coordinated; rapid
e coli dna replication begin at a unique site
-Replication begins in E. coli at a unique 245 base pair site called the __.

-__ protein binds at distinct sites in the locus and then recruits __, a helicase that uses ATP hydrolysis to unwind the locus.
- oriC locus
- DnaA; DnaB
e coli dna replication begin at a unique site
-__ bind to the __-rich sites in the locus to prevent reannealing of the helix.

-The resulting complex of DNA and proteins is called the __
- single stranded binding protein (ssb); AT
- prepriming complex
- dna polymerase cannot initiate new __
--unable to covalently link 2 individual __ together
-- able to covalently link a __ to a __
- strands
-- nucleotides
-- nucleotide; primer
__ Synthesized by __ Enables DNA Synthesis to Begin
-A special type of __ called __ synthesizes short RNA (≈10 nucleotides) complementary to a DNA strand that then serves as a __ for DNA synthesis.
Rna primer; primase
- rna polymerase; primase; primer
-Primase is part of a large complex called the __
-__, employing a third enzymatic activity, a __ activity, subsequently removes the primers and replaces the RNA with __.
- primasome
- dna polymerase I; 5>3 exonuclease; dna
One Strand of DNA is Made __ while the Other Strand is Made in __
-The site of replication is called the __.

-The fork moves in one direction, so both strands are copied __.

-However, all DNA polymerases synthesize DNA only in the __ direction.
continuously; fragments
- replication fork
- simultaneously
- 5>3
-At the replication fork, the __ strand is synthesized continuously in the __ direction.

-The __ strand is synthesized discontinuously as small pieces called __ in the __ direction.
- leading; 5>3
- lagging; okazaki fragments; 5>3
__ begins each okazaki fragment
-__ is a complex containing __ enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)
-__ uses the __ to start the lagging-strand DNA synthesis
rna primer
- primosome; primase
- dna polymerase; rna primer
- The holoenzyme responsible for the majority of DNA synthesis in E. coli is __, a multi-protein complex.

-__ is highly processive, once it begins catalysis it rarely releases the __.
- dna polymerase III
- dna polymerase III; dna substrate
-The source of the processivity is the __, a dimer that encircles the helix.
-The sliding clamp is loading onto the DNA by a protein called the __, which uses the energy of __ to open and close the clamp
- sliding clamp (Beta 2 subunit)
- clamp loader; atp hydrolysis
-The __ strand is looped so that it passes through the polymerase active site in the __ direction, allowing synthesis to occur in the __ direction
-__ binds the single stranded DNA - replication does not result in large patches of open __.
- lagging;3>5; 5>3
- ssb; ssDNA
as replication proceeds:
-__ unwinds DNA template
-replication continues on __ strand
-__ makes a new primer on the __ strand
-__ strand core __ finishes an __
- helicase
- leading
- primase; lagging
- lagging; polymerase; okasaki fragment
-Lagging strand polymerase encounters preceding Okasaki fragment, releases the lagging strand. Nick between Okasaki fragments will be sealed by __ and __
-Polymerase core on __ strand never releases - keeps replication processive
- polymerase I; dna ligase
- leading
Lagging strand polymerase then binds newly made __ - starts next Okasaki fragment
Okazaki Fragments Are Joined by Action of __ and __
-Okazaki fragments are joined to produce a continuous strand of DNA in 3 steps:
(1) Removal of the __ by The __ activity of __
(2) Synthesis of replacement DNA, __: polymerase activity of pol I synthesizes DNA in place of RNA
(3) Sealing of adjacent DNA fragments by __
dna poly I; dna ligase
- rna primer; 5>3 exonuclease; dna pol 1
- nick translation
- dna ligase
Problem: after Okazaki fragment synthesis is complete, have gap between end of Okasaki fragment and RNA primer
-__ extends the Okasaki fragment while its __ activity removes the __. This is called __.
-__ then dissociates, __ binds to the nick.
-__ then catalyzes the formation of a __ linkage between 3’-hydroxyl and 5’-phosphate of adjacent Okazaki fragments - enzyme then dissociates from DNA
- dna pol I; 5>3 exonuclease; rna primer; nick translation
- dna pol I; dna ligase
- dna ligase; phosphodiester
-__ chromosomes are large linear, double-stranded DNA molecules
-Rate of __ slower than in E. coli but multiple __ enables rapid replication of genome
- eukaryotic
- replication; origins
-Multiple __ of replication are required with each origin of replication representing a replication unit or replicon. __ have ≈ 30,000 replicons.

-Proteins called __ allow only one replication per replicon per round of DNA synthesis.

-__, which displays primase activity, initiates DNA replication in eukaryotes, generating a DNA molecule ≈ 20 dNTP in length.

-__, a more processive enzyme, extends the chains. The switch from polymerase __ to __ is called polymerase switching.
- origins; humans
- licensing factors
- dna polymerase alpha
- dna polymerase delta; alpha; delta
__: unique structures at the ends of linear chromosomes
__, the free end of linear dna, have two problems
1. they are susceptible to damage by __
2. due to the nature of dna synthesis, one strand will __ upon each round of dna synthesis
1. nucleases
2. shorten
__: protecting the end from degradation
- the end of the __ strand is rich in __, and contains hundreds of tandem repeats
- the stretch of __ rich single strand dna can form a loop structure to protect the end of a chromosome
- leading; guanine
- guanine
__ replicates telomeres
-__ contains an __ that it uses to extend the leading strand.

-This prevents the lagging strand from getting progressively shorter

-In rapidly dividing cells, including __, telomeres must be maintained by the telomerase to prevent shortening of the __, which would lead to cell death.

-High __ activity is a characteristic of cancer cells
- telomerase; rna template
- cancer cells; lagging
- telomerase
__: an enzyme that can use RNA as a template to synthesize DNA
-present in retroviruses, such as __
-also called __
-high error rate because it has no __ capabilities
-target of __ drugs
reverse transcriptase
- hiv
- rna directed dna polymerase
- proof-reading
- antiviral
-DNA is the only cellular macromolecule that can be __ - __ can have a very high cost
-DNA damage includes: __ modifications, __ deltions or insertions, __ of dna strands, breakage of __
-DNA damage can result from __ (chemicals, radiation) and from __ made during normal cellular processes
- repaired; mutations
- base; nucleotide; cross linking; phosphodiester bonds
- environmental agents; errors
-The simplest source of DNA damage is the incorporation of an incorrect base during __ that escapes proofreading by the __.

-Other errors include insertions, deletions or breaks in one or both strands, which may halt DNA synthesis altogether.

-Special DNA polymerases, called __, can replicate across the damage and generate a rough draft of the damaged sequence. These are not as precise as replicative polymerases
- replication; dna polymerases
- translesion polymerases
-Some sequences, such as __ regions, are inherently prone to errors

-Some genetic diseases such as Huntington’s Disease are related to expansion of these regions. __ is repeated up to 31 times in unaffected people, much more in those with the disease
-Formation of alternative structures during replication leads to the expansion
- triplet repeat
- huntington's disease; CAG (encoding glutamine)
- bases can be damaged by __, __, and __
-Chemicals that alter specific bases after replication is complete are called __.

-Hydroxyl radicals oxidize __ to __, which base pairs with __ instead of __ during the next round of replication.
-oxidizing agents; alkylating agents; light
- guanine; 8-oxoguanine; adenine; cytosine
-Deamination can be mutagenic. __ can be deaminated, forming __, which pairs with __ instead of __
- adenine; hypoxanthine; cytosine; thymidine
-Bases can react with hydrocarbons in an __ reaction.

-__ is converted into a highly reactive epoxide that reacts with __ forming a compound that, during replication, converts a __ base pair into a __ base pair.

-Chemicals in cigarette smoke and automobile exhaust can also be converted into reactive __
-Ultraviolet radiation covalently links adjacent __ thereby blocking replication.
- alkylation reaction
- aflatoxin; guanine; g-c; a-t
- epoxides
- pyrimidines
DNA repair systems follow the same mechanistic outline:

1.__ the inappropriate base(s).

2.__ the inappropriate base(s).

3.Fill in the resulting gap with DNA __ and __.

-The first DNA repair mechanism occurs when __ proofreads the newly synthesized DNA and correct mismatches.
1. recognize
2. remove
3. dna poly I; dna ligase
- dna polymerase
-Some damage can be corrected with direct repair
-T-T dimers can be directly repaired by many organisms (but not __ or other placental mammals)
-Repair of thymine dimers by __ - __
- humans
- dna photolyase; photoreactivation
-__ correct errors not corrected by proofreading that cannot be directly repaired

-In E. coli, need one protein to recognize the error (__), one to recruit an exonuclease to cleave the DNA (__), and the exonuclease (__).

-Parent strand may be “marked” by __ of adenines so that repair machinery can recognize the correct base
- mismatch repair systems
- mutS; MutL; MutH
- methylation
-__ uses several enzymes to correct modified bases.

-__ such as AlkA remove the base. The site of the missing base is called an __.

-The DNA backbone at the AP site is cleaved by __ and a __ excises the deoxyribose phosphate.

-The gap is repaired by __ and __
- base-excision repair
- glycosylases; ap site
- ap endonuclease; phosphodiesterase
- dna poly I; dna ligase
-Hydrolytic deamination of __ to __ is one of most common types of DNA damage
-Uracil in place of cytosine causes incorporation of an incorrect base during replication
-Corrected by __
-__ hydrolyze base-sugar N-glycosidic bonds
-__ bases are then removed and replaced
- cytosine; uracil
- base excision repair
- dna glycosylases
- deaminated
-Use of __ in DNA preserves the integrity of the genetic information.

-If uracil naturally occurred as a base in DNA, __ would lead an A-U pair replacing a G-C pair after the next round of replication.

-Use of __ instead of __ allows the detection of deamination of __.

-If uracil is detected in DNA, it is removed by __ and the resulting AP is repaired with the insertion of __.
- thymidine
- cytosine deamination
- thymidine; uracil; cytosine
- uracil dna glycosylase; cytosine
-If base-excision fails to recognize the damaged base, the mutation may be corrected by the __ system.

-An enzyme complex recognizes the distortion of the DNA caused by offending base. The __ cleaves the DNA at two sites, several nucleotides on each side of the distortion.

-__ and __ close the resulting gap
- nucleotide-excision repair
- UvrABC excinuclease
- dna poly I; dna ligase
what happens when excision repair does not function?
-__– genetic disorder in DNA nucleotide excision repair genes – a number of different proteins involved
-Patients have extreme sensitivity to the sun, much greater risk of skin cancer, and about 20% have neurological disorders
- xeroderma pigmentosum (xp)
-__ are caused by mutations in genes that control growth.

-__ act a tumor suppressors.

-If both copies of a DNA repair enzyme are mutated, __ is more likely to develop

-Because tumors lack DNA repair systems, damaging the DNA with chemicals such as __ and __ is a strategy to prevent cancer growth.
- dna repair enzymes
- cancer
- cyclophosphamide; cisplatin
-The __ test - simple, sensitive means of determining if a chemical is mutagenic.

-Example: bacteria that require histidine because of a mutation in its biosynthetic pathway are treated with the chemicals. Then grow the bacteria without the required amino acid. Bacterial growth (appearance of colonies) suggest that the chemical caused a mutation which restored histidine synthesis
- ames
-__ - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome
-__ - occurs between pieces of DNA that have closely related sequences (e.g. exchange between paired chromosomes during meiosis). Genetic recombination is a type of homologous recombination.
-__ occurs between unrelated sequences (e.g. Transposons )
-__ - recombination at a specific location
- homologous recombination
- nonhomologous recombination
- site specific recombination
why is recombination needed?
-__ gives new genetic variation

- __ creates different combinations of mutations within a genome - parents chromosomes are not the same as progeny chromosomes
of DNA
- mutation
- recombination
-__ probably evolved to help DNA repair (confers a selective advantage)
-Severe __ in DNA can be repaired by strand exchange from intact daughter chromosome to defective daughter
-Very important process - more than 100 genes from E. coli are involved in recombination and repair
-recombination enzymes
- lesions
Recombination is a complicated process – many proteins!
1.Double strand break is recognized,the __ ends are digested, single-strand regions are bound by __.

2.__ occurs - single strand from damaged DNA replaces a strand in undamaged DNA. This three-stranded structure is called a __.

3.__ occurs, using the undamaged DNA as a template.

4.A second strand invasion occurs to complete the repair, forming a structure called a __.

5.Cleavage and ligation yield two intact __
1. 5'; rad51
2. strand invasion; displacement loop
3. dna synthesis
4. holliday junction
5. helices
what happens when ds break repair doesn't function?
-__ and __ both required for normal recombinational repair of ds breaks in DNA
-form complex with __ (eukaryotic equivalent of RecA)
-approx. 12,000 cases of breast cancer a year in north America are related to mutations in these two proteins
- brca1; brca2
- rad51
-BRC repeats of __ interact directly with __

-__ may serve to deliver __ to the site of DNA damage

-Conserved __ appears to bind ssDNA and dsDNA
-brca2; rad51
- brca2; rad51
- c-terminus
The synthesis of RNA from a DNA template is called transcription, a process catalyzed by __
rna polymerase
RNA polymerase has the following requirements:

1.A __. The sequence of the newly synthesized RNA is complementary to the DNA template. The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the __.
Activated precursors in the form of the

2.four __

3.Divalent metal ions, usually __ or __
1. template; coding strand
2. ribonucleoside triphosphates (atp, gtp, utp, ctp)
3. Mg+2; Mn+2
RNA polymerase initiates and elongates the RNA product, with the chain growing in the __ direction. The 3’OH of the growing chain attacks the inner most __ of the incoming ribonucleoside triphosphate.
-5>3; phosphoryl (alpha) group
A __ is a transcriptional unit. Types of RNA made are:
(1) __
--Carries amino acids to translation machinery
--Very stable molecules
(2) __
--Makes up much of the ribosome
--Very stable, majority of cellular RNA
(3) __
--Encodes message from DNA to ribosomes
--Rapidly degraded by __
--Small fraction of steady-state RNA, but use a large percentage of synthetic capacity of cell
(4) __
-- participate in metabolic events; many have catalytic activity
1. tRNA
2. rRNA
3. mRNA
-- nucleases
4. small RNA
E. Coli RNA Polymerase Is an Oligomeric Protein
-Core enzyme: __ participates in many of the transcription reactions
-The holoenzyme, consisting of __ subunits, initiates RNA synthesis.
-The core enzyme, composed of ___ subunits, elongates the RNA product.
-__: role in initiation of transcription
- a2bbw
- sigma subunit
-The three stages of RNA synthesis are __, __, and __.

-How does the RNA polymerase know where to start? Specific __ sequences called __ direct __ to the proper initiation site.

-In E. coli, two DNA sequences that act as a promoter for many genes are the __ and the __.
- initiation; elongation; termination
- dna; promoters; rna polymerase
- -10 sequence (pribnow box); -35 sequence
-__ will closely match the consensus sequence
-__ tend to have multiple substitutions at these sites
-Other sequences __ of the promoter and the action of transcription factors can enhance promoter effectiveness.
- strong promoters
- weak promoters
- upstream
-The __ of prokaryotic RNA polymerases is required for promoter recognition and formation of the complex
-s Subunit increases the affinity of the __ for specific __ sequences
-s subunit also decreases the affinity of the __ for __ regions
-Core polymerase (no s subunit) binds __ nonspecifically
- sigma subunit
- core polymerase; promoter
- core polymerase; nonpromoter
- dna
-The s factor acts __ - one s can aid many __ in finding promoters

-E. Coli has many different s factors that recognize different consensus sequences at the promoter
- catalytically; polymerases
-__ rapidly searches for and binds promoter
- Unwinding of DNA at the initiation site requires a __ – often the rate limiting step
-RNA pol (R) and promoter (P) shift from: __ to __
- rna polymerase
- conformational change
- (RPc) closed complex (dsDNA); (RPo) open complex (17 bp unwound)
-__ begins transcription
-Once the DNA is unwound, __ can take place.

-__ can start RNA synthesis de novo – no primer needed!
- rna polymerase
- elongation
- rna polymerase
- elongation takes place at __
- The region containing the RNA polymerase, DNA, and the RNA product is called the __.

-The transcription bubble moves along the DNA as DNA is unwound and then rewound, while the RNA product is extruded from the complex. A __ helix of approximately __ is an intermediate in RNA synthesis
- transcription bubbles
- transcription bubble
- dna-rna hybrid helix; 8 nucleotides
-Transcription continues until a __ is reached
-Transcription complexes disassemble at the __ end of genes at specific __
-Two types of termination sequences
1) __ - generally associated with a region of the RNA transcript that forms a hairpin structure, with a series of __
- termination signal
- 3'; termination sequences
1) unstable elongation complex; u residues
-__ pauses after it has synthesized RNA that folds into a __
-__ with __ is unstable – __ is the weakest of the Watson-Crick base pairs
-Pause caused by __ permits weak RNA-DNA hybrid to __
- rna polymerase; hairpin
- RNA-DNA hybrid; oligo(u); rU-dA
- hairpin; dissociate
- __ dependent termination sites
-__ triggers disassembly of the transcription complex at some pause sites
-__ binds to __ chain, destabilizing the RNA-DNA hybrid and terminating transcription
-A hexamer of rho binds a __ stretch on RNA
-Note that both types of termination involve __ on the newly synthesized RNA
- rho
- rho
- rho; ssRNA
- 72 nt
- signals
-Precursors of __ and __ are cleaved and chemically modified after transcription
-Although __ undergoes little or no modification after synthesis in bacteria, __ or __ are modified:

1. The final mature __ is cleaved from a larger precursor molecule.
2.Many __ transcripts lack __ sequence at the __ end of the strand. These nucleotides are added post-transcriptionally.
3.The bases and riboses of tRNA and rRNA are modified, for instance, by the attachment of __.
- tRNA; rRNA
- mRNA; rRNA; tRNA
1. RNA
2. tRNA; CCA; 3'
3. methyl groups
-The antibiotics __ and __ inhibit transcription, but by different mechanisms.

-__ inhibits initiation by binding to the polymerase and blocking the exit of the nascent RNA.

-__ intercalates between the bases of the DNA double helix, preventing the DNA from being used as a template
- rifampicin; actinomycin
- rifampicin
- actinomycin
-The __, an Example of Negative and Positive Regulation
-E. coli can use __ and other __ as a carbon source when __
-Uptake and catabolism of __ requires three proteins:
- lac operon
- lactose; beta galactosides; glucose is absent
- beta-galactosides
1. lactose permease (lac Y)
2. beta galactosidase (lac Z)
3. thiogalactoside transacetylase (lac A)
(1) Lactose permease (lacY) - __ for uptake of __
(2) b-Galactosidase (lacZ) - hydrolyzes __ to __
(3) Thiogalactoside transacetylase (lacA): __ nonmetabolizable __
1. transporter; beta galactosides
2. beta galactosides; hexoses
3. acetylates; beta galactosides
-When glucose is __ these proteins are synthesized in limited amounts
-When glucose levels are __ these proteins are synthesized in larger amounts if __ are present
- present
- low; beta galactosides
-Three genes - lacZ, lacY and lacA form an __ that is transcribed from a single __
-Resulting large __ molecule contains three separate protein-coding regions (3 genes)
-operons with multiple protein-coding regions are common in __ but not __, though they are used in C. elegans
- operon; promoter (Plac promoter)
- mRNA
- prokaryotes; eukaryotes
- __ Blocks Transcription
-lac Operon expression is controlled by the __ - __ is encoded by the __ (under control of __), located upstream from the lac operon
-lac Repressor binds simultaneously to __
-__ is adjacent to the promoter, __ is within the coding region of lacZ
- lac repressor
- lac repressor protein; lac repressor; lacl gene; (p promoter)
- 2 repressor binding sites (operators)
- operator (O1); (O2)
-The __ binds simultaneously to both operators O1 and O2
-This causes the DNA to form a __ which blocks initiation of transcription
-__ act as inducers to cause the repressor to dissociate from the operon allowing __ to continue
- lac repressor
- stable loop
- beta galactosides; transcription
- __: inducer for lac operon
-Several __ can act as inducers to start gene transcription
-In the presence of lactose, __ is the inducer, binds tightly to __ and causes a conformational change that decreases affinity of lac repressor for __
-b-Galactosidase converts __ to __
- allolactose
- beta-galactosides
- allolactose; lac repressor; operator
- lactose; allolactose
-__ Regulatory Protein Activates Transcription
-The lac operon is maximally transcribed when __ are the only carbon source
-When __ is present transcription is reduced 50-fold, (catabolite repression)
-A weak __ in these operons is promoted to a stronger one by an activator (in the absence of glucose)
-cAMP regulatory (or receptor) protein (__) is the activator - __ is also known as __
-cAMP levels increase when __
- cAMP
- beta galactosides
- glucose
- promoter
- (crp); CRP; catabolite activator protein (CAP)
- glucose decreases
-Some __ can sense environmental signals as well as encode proteins.

-These mRNA have special structures, called __, that bind small molecules that cause a structural change in the __ that terminates the synthesis of the mRNA.
- bacterial mRNA
- riboswitches; riboswitch
-The basics of RNA synthesis are the same for __ and __

-__ organisms use differential gene regulation to generate different cell types.
- eukaryotes; prokaryotes
- multicellular
Gene expression in eukaryotes is influenced by three important characteristics:

1. __ regulation

2.__ processing, including extensive processing of __ precursors

3.The __, which separates the site of __ synthesis from that of __ synthesis
1. complex transcription
2. RNA; mRNA
3. nuclear membrane; RNA; protein
- eukaryotic cells have three __
-RNA synthesis is catalyzed by three __ that differ in DNA substrate specificity, location, and sensitivity to the toxin α-amanitin.

-All the polymerases are similar in structure, but RNA polymerase II has a unique domain, called the __, that plays an important regulatory role
- rna polymerases
- rna polymerases
- carboxyl terminal domain
-RNA poly I: location is __; transcripts include __, __, __; effect on alpha amanitin: __
-RNA poly II: location is __; transcripts include __ and __; effect on alpha amanitin: __
-RNA poly III: location is __; transcripts include __ and __; effect on alpha amanitin: __
- nucleolus; 18s, 5.8s, 28sRNA; insensitive
- nucleoplasm; mRNA and snRNA; strongly inhibited
- nucleoplasm; tRNA and 5sRNA; inhibited by high []
-Eukaryotic promoters, also called __, are more complicated than bacterial promoters. Each type of polymerase has distinct promoters.

-The promoters bind to proteins, called __ or __, that regulate polymerase activity.
- cis-acting elements
- trans-acting elements; transcription factors
RNA polymerase II
-Transcribes all __ genes and some small __ encoding genes
-Protein-encoding RNA synthesized by RNA pol II is called __
-20%-40% of all cellular __
-General ___ interact directly with RNA pol II and control initiation at class II genes
-Elaborate regulation of RNA pol II accounts for cell differentiation and development.
- protein-encoding; rna
- mRNA precursor (or hnRNA)
- rna synthesis
- transcription factors
-Most genes transcribed by RNA pol II have an A/T rich sequence called __ located -24 to -32
-Other important sequences include the __ at the transcriptional start site and a __ at +28 to +32
-Many promoters also have __ or __ boxes upstream
- tata box
- initiator element; downstream core promoter element
- caat; gc
-The __ complex initiates assembly of the active transcription complex
- Transcription factors of RNA poly II= __
-In genes with a TATA box, the __, a component of TFIID, binds to the TATA box. Other transcription factors bind to generate the basal transcription complex.
-__ binds to the complex and phosphorylates the __ of the polymerase, facilitating the transition from initiation phase to the elongation phase.
- tata binding protein (TBP)
- TFIIH; carboxyl terminal domain
-Enhancer sequences are __ that have no promoter activity but can stimulate the effectiveness of promoters even when located thousands of nucleotides from the start site.

-Inappropriate enhancer use may cause __

-Chromosomal translocations can place a gene under the control of a powerful enhancer. For instance dysregulation of the gene myc, a transcription factor, resulting from the translocation of an enhancer to a region near the myc gene plays a role in the development of __ and __
- cis-acting elements
- cancer
- burkitts lymphoma; b-cell leukemia
-Many other proteins play a role in regulating the efficiency and specificity of gene transcription

-A large complex called __ acts as a bridge between enhancer-bound activators and proteins, including the polymerase, at the promoter.

-Effects on transcription can
depend on the other components
of the regulatory complex
(combinatorial control).
- mediator
- gene expression is regulated by __
-Gene expression in eukaryotes must respond both to conditions within the __ and to __

-__ hormones are one class of regulatory molecules that control gene expression.

-__ controls the genes in the development of female secondary sex characteristics. It exerts its effects by forming a complex with a specific receptor protein called the estrogen receptor.
- hormones
- cell; external stimuli
- steroid
- estradiol
-__ have similar domain structures
-The estrogen receptor is part of a larger class of regulatory proteins called __, all of which are activated by binding of small molecules or ligands.

-__ (previous) bind to specific regions of the DNA called __. Thus, the estrogen receptor binds to the estradiol response element (ERE).
- nuclear hormone receptors
- nuclear hormone receptors
- nuclear hormone receptors; response elements
Nuclear hormone receptors have two conserved domains.

1.The __ has zinc-finger domains that confer specific DNA binding.

2.Ligand binding to the
causes a structural change
that enables the receptor
to recruit other proteins to
regulate transcription.
1. dna binding domain
2. ligand binding domain
-__ leads to structural rearrangements in the receptor.

-These changes do NOT affect binding to the __

-Instead, the changes
promote recruitment of
additional proteins that
assist in __
- ligand binding
- response element
- transcription
- __ and __ are recruited by nuclear hormone receptors
-Proteins that bind the receptor after ligand binding are called __.

-Some nuclear hormone receptors repress transcription in the absence of ligand. __ proteins bind to them when ligand is absent to assist in repression of transcription.
- coactivators; corepressors
- coactivators
- corepressor
- __ receptors are targets for drugs
-Ligands that activate a nuclear hormone receptor are called __, while ligands that inhibit the receptor are called __.

-Some __ require the estradiol-receptor complex, and growth of these cancers can be slowed by administering receptor antagonists, such as __ and __

-Such antagonists to the
estrogen receptor are
called __.
- steroid hormones
- agonists; antagonists
- cancers; tamoxifen; raloxifene
- selective estrogen receptors modulators (SERMS)
-__ results in chromatin remodeling
-How do coactivators stimulate transcription? One mechanism involves loosening the interaction between histones and DNA, so the DNA is more accessible to transcription machinery.

-A common means of weakening the interaction of the histones with the DNA is by __ of the __ on specific __ residues.
- histone acetylation
- acetylation; histones; lysine
-__ is also important for transcription!
-__ located in the nucleus generates acetyl CoA that is used by __ to modify histones.

-__ are components of
coactivators or are
recruited by coactivators.

-The __ are
extended and fit in the HAT
active site.
- acetyl CoA
- atp-citrate lyase; histone acetyltransferases (HATS)
- HATs
- histone tails
1. Acetylation reduces the affinity of __ for __.

2. __ residues are interaction sites for many proteins which regulate transcription, including factors which bind the __

3. __ residues also bind and recruit chromatin remodeling proteins.
1. histones; dna
2. histone acetyllysine; tata-box
3. acetyllysine
-The __ of histones is reversible, allowing genes to be expressed or repressed at the appropriate time.

-Histone __ catalyze the removal of acetyl groups, leading to __

-Histones can also be __ or __

-All covalent
of histone are
- acetylation
- deacetylases; inhibition of transcription
- methylated; phosphorylated
RNA processing in eukaryotes
-Large precursor RNA (__) is processed to __, __, and __, which are components of the ribosome.

-Before cleavage, some bases and riboses are modified in a process catalyzed by RNA-protein complexes called __
- (45s); 18s; 28s; 5.8S rRNAs
- small nucleolar ribonucleoproteins (snoRNP)
Transfer RNA is extensively processed
-__ removes nucleotides from the 5’ end,while the __ adds nucleotides to the 3’ end.

-Bases and riboses are also __.

-Many eukaryotic tRNA
precursors contain an
__ that is removed by
an endonuclease, and
the resulting products
are joined by a ligase
- RNase P; CCA adding enzyme
- modified
- intron
-All eukaryotic __ undergo modifications to increase their stability and make them better substrates for translation
-Ends are modified so they are no longer susceptible to __
-The __ ends are modified (capped) before the mRNA precursors are completely synthesized
-Addition of __ required for splicing and other processing events. __ is also site where ribosomes bind.
- mRNA precursors
- exonuclease degradation
- 5'
- cap; cap
-Formation of a cap at the __ end of a eukaryotic mRNA precursor involves an unusual __
1. __ removes phosphate at 5' end
2. 5' end gets __ group from __, catalyzed by __, product called a cap
- 5'; 5'-5' triphosphate linkage
1. phosphohydrolase
2. gmp; gtp; guanalyltransferase
- the caps can also undergo __
3. __ base is methylated
4. __ groups may also be methylated
- methylation
3. n-7
4. 2'-hydroxyl
- __ at the 3' end of mRNA precursors
-Eukaryotic mRNA precursors are also modified at their 3’ ends - __ adds up to 250 adenylate residues to the 3’ end
-This poly A tail is progressively shortened by __
-The __ increases the time required for
nucleases to reach the coding region
- poly a tails
- poly a polymerase
- 3' exonucleases
- poly a tail
-Most genes in eukaryotes consist of __ (coding regions) and __ (noncoding regions). Introns vary in length from 50 to 10,000 nucleotides.

-The __ are joined by splicing
- exons; introns
- exons
intron-exon junctions have several common features:
1.The __ end of the junction has the sequence 5’AGGUAAGU 3’ with the first __ from the 5’ end marking the beginning of the __.

2.The 3’ end of the intron is marked by a stretch of __ followed by any base, a C, and then the intron ends with __.

3.The branch site is located 20-50 nucleotides from the __ end of the intron.
1. 5'; GU; intron
2. pyrimidines (polypyrimidine tract); ag
3. 3'
-Splicing is facilitated by __ which associate with the pre-mRNA to form the splicing apparatus called the __.

-__ at the 5’ splice site, followed by __ binding at the branch site.

-Binding of __ completes splicesome formation.
- snRNPs; spliceosome
- u1 snRNP; u2 snRNP
- u4-u5-u6 tri-snRNP
-The __ and the __ are the actual catalysts of splicing.

-The catalytic snRNAs facilitate __ reactions that remove the introns and join the exons.
- u2 snRNA; u6 snRNA
- transesterification
-__ in either pre-mRNA or splicing factors can result in pathological conditions.

-Defects in splicing or alternative splicing may cause up to 15% of all genetic diseases.

-__, a
disease of acquired
blindness, is due to a
mutation in the
U4-U5-U6 tri-snRNP.
- mutations
- retinitis pigmentosa
Some __, diseases resulting from defective hemoglobin synthesis, are caused by mutations at splice sites in the pre-mRNA for the β chain of hemoglobin.
-Introns allow formation of alternative forms of a protein called __
-__ is a powerful mechanism for expanding protein diversity.

-In alternative splicing, a __ can be spliced in different patterns, generating proteins with different functions.
- splice variants
- alternative splicing
- pre-mRNA
what controls how splicing sites are selected
-__ splicing factors bind to __ sequences in the pre-mRNA

-__ leads to different coding sequences, and proteins with different functions
- trans-acting; cis acting
- alternative splicing
-Transcription and splicing are coordinated by the __ of RNA polymerase II.

Functions of the CTD include:

1.Recruiting __ to synthesize the 5’ cap.

2.Recruiting components of the __

3.Recruiting an __ that cleaves the pre-mRNA to expose the site for __.
- carboxyl-terminal domain (CTD)
1. enzymes
2. splicing complex
3. endonuclease; poly a addition
-The coding information of some mRNAs can be altered by __.

-The unedited transcript forapolipoprotein B, a protein important in lipid transport, encodes a 512 kd protein,apo B-100.

-RNA editing deaminates a cytidine in the codon for glutamine forming uridine, which generates a stop codon.The edited transcript encodes for apo B-48, a 240 kd protein
- RNA editing
-Some RNAs, called __, function as catalysts.

-Introns from certain organisms can self-splice. Self-splicing or __ were initially identified in rRNA from Tetrahymena.

-Group 1 introns
require __
as a cofactor.
- ribozymes
- group 1 introns
- guanosine
the genetic code
-__ – four letter nucleotide “alphabet” is translated into the 20-letter amino acid “alphabet”
-__ – three nucleotides which specify an amino acid. A one letter or two letter genetic code made from four bases would not have been sufficient to encode for 20 amino acids.
-__ - each potential starting point for interpreting the 3 letter code. A nonoverlapping code is used, so the reading frame is very important
-__ - the sequence of bases is read sequentially from a fixed starting point until termination in a 5’ to 3’ direction.
- translation
- codons
- reading frame
- directionality
-the genetic code is a __ code
- translation of the correct message requires selection of the correct __
- reading frame
-almost all living organisms use the same genetic code
1.The genetic code is __. In any organism each codon corresponds to only one amino acid.
2. There are multiple codons for most amino acids (code is __), and synonymous codons specify the same __
3. The first two nucleotides of a codon are often enough to specify a given __
4. codons with similar sequences specify similar __
5. only __ of the 64 codons specify amino acids
1. unambiguous
2. (degenerate); synonymous codons
3. amino acid
4. amino acids
5. 61
- the termination codons are as follows:
- the initiation codon is __, specifies the amino acid __, and is the initiation site for protein synthesis
- uaa, uga, uag
- aug; methionine
- the genetic code is nearly __
- __ also use variations of the genetic code
- universal
- mitochondria
-__ function as an adaptor molecule between a codon and an amino acid.

-There is at least one tRNA molecule for each __.

General characteristics of tRNA molecules include:
1.Each is a single strand of RNA between __ and __ amino acids in length.

2.The three-dimensional structure of the molecule is __.
3. In a two-dimensional representation, all tRNA molecules appear as a __ pattern. The amino acid–accepting region is the __, which contains the 3’ __ terminal region. Many of the nucleotides are involved in hydrogen bonds that form stems and loops.
4.Transfer RNA molecules contain unusual bases, such as __, or bases that have been modified.
5.The 5’ end is __ and the 5’ terminal residue is usually __.
6. The amino acid is attached to a __ of __ in the __ region of the acceptor stem.
7. The __ is in a loop near the center of the sequence.
- tRNA
- amino acid
1. 73; 93
2. L-shaped
3. cloverleaf; acceptor stem; cca
4. inosine
5. phosphorylated; pG
6. hydroxyl group; adenosine; cca
7. anticodon
-Some transfer RNAs recognize more than one codon due to __ in base pairing
1.Codons that differ in either of the __ must be recognized by different tRNA.

2.The first base of the
anticodon the degree
of wobble. If the first
base is __, the
anticodon can recognize three codons
- wobble
1. first two nucleotides
2. inosine
synthesis of long proteins requires a low __
error frequency
- amino acids are activated by attachment to __
-To be incorporated into proteins, amino acids must be activated.

-Amino acids are activated by formation of an __ linkage between the carboxyl group of the amino acid and either the 2’ or 3’ hydroxyl group of the terminal adenosine of the tRNA, forming an aminoacyl tRNA or __
- tRNA
- ester; charged tRNA
-___ amino acids are covalently attached to the 3’ end of each tRNA molecule (named as: alanyl-tRNAAla)
-__ catalyze these reactions
-Most species have at least __ different aminoacyl-tRNA synthetases (1 per amino acid)
-__ are high-energy molecules (the amino acid has been “activated”)
-The activation of an amino acid by aminoacyl-tRNA synthetase requires __, both high energy phosphate bonds are broken
- aminoacyl tRNA
- aminoacyl tRNA synthetases
- 20
- aminoacyl tRNAs
- atp
- amino acids are first activated by __
- the first step is the formation of __ or __
- the aminoacyl group is then transferred to a specific tRNA recognized by the __
- the aminoacyl amp never leaves the active site of the __
- adenylation
- aminoacyl adenylate; aminoacyl amp
- synthetase
- synthetase
-Attachment of the correct amino acid to the corresponding tRNA is a critical step
-__ binds ATP and the correct amino acid (based on size, charge, hydrophobicity)
-__ then selectively binds specific tRNA molecule based on structural features
- synthetase
- synthetase
- some aa-tRNA synthetases can be __
-Synthetases are the true translators of the genetic code in that they assign a particular __ to a specific __.

-Many aspects of the tRNA molecule, in addition to the anticodon, are used as recognition sites by the __ to achieve this specificity.
- amino acid; tRNA
- synthetases
-The __ is the site of protein synthesis.

-In E. coli, the ribosome sediments at 70S and is made of two subunits, the large __ subunit and small __ subunit.

-The 50S subunit = __

-The 30S subunit = __.
- ribosome
- 50s; 30s
- 34 proteins + 23s RNA+ 5S RNA
- 21 proteins + 16S RNA
-Two-thirds of the mass of ribosomes is __, which is critical for the structure and function of the ribosome.

-The ribosomal RNAs fold into complex structures with many short duplex regions.

-Ribosomal RNA is the actual catalyst for __, with the ribosomal proteins making only a minor contribution.
- protein synthesis
-Because transcription and translation both occur in the __ direction, bacterial protein synthesis begins before transcription is complete.

-Several ribosomes can translate an mRNA molecule at the same time, forming __ or __
- 5>3
- polyribosomes; polysomes
- protein synthesis decodes the info in __
- protein synthesis is very ___
- protein synthesis consists of three parts: __, __, and __
-Initiation requires the cooperation of __, __, __, and proteins called __. – very complicated.
- mRNA
- energetically costly
- intiation, elongation, and termination
- ribosomes, tRNA, mRNA, initiation factors
- Ribosomes have three tRNA binding sites
1.The __ binds the incoming tRNA.

2.The __ binds the tRNA with the growing peptide chain.

3.The __ binds the uncharged tRNA before it leaves the ribosome.
1. A (aminoacyl) site
2. P (peptidyl) site
3. E (exit) site
-The __ holds the mRNA

-Each tRNA is in contact with both the __ and __ ribosomal subunits.

-The acceptor end of the tRNAs
in the __ and __ are near
one another – this is where the
__ is formed. A
channel connects this site to
the back of the ribosome
through which the peptide exits the ribosome during synthesis.
- 30S ribosomal subunit
- 30S; 50S
- A site; P site; peptide bond
-In bacteria, the start signal is __ preceded by several bases that pair with __
-Many mRNAs in bacteria are __ - a single mRNA encodes for multiple proteins. Each of the coding regions has its own __.

-The first codon to be translated is usually __
-Initiation in bacteria begins at least 25 nucleotides from the __ end of the mRNA.
- AUG (or GUG); 16S ribosomal RNA
-polycistronic; initiation site
- AUG (methionine)
- 5'
-The nucleotides between the 5’ end of the mRNA and the first codon translated are called the __
-The UTR contains a __ that directs protein synthesis machinery to the start site. The previous sequence is __, about 10 bp upstream of the start site, and interacts with the
__ to correctly position
the AUG codon in
the __.
- untranslated region (UTR)
- shine dalgarno; purine rich; 16S rRNA; A site
- bacterial protein synthesis is initiated by __
-A modified form of methionine, __, is the initiator amino acid in most proteins in bacteria.

-It is attached to the __. __ binds only to the initiation codon (AUG- not to other AUGs.

-Another tRNA—__—recognizes internal codons for methionine.

-The same __ activates both tRNAm and tRNAf and a specific __ modifies the methionine attached to tRNAf
- formylmethionyl tRNA
- n-formylmethionine (fMET)
- initiatior tRNA (tRNAf); fmet-tRNAf
- tRNAm
- synthetase; transformylase
-f-Met-tRNAf is placed in the __ during formation of the __ initiation complex
-__ assist in the assembly of the protein-synthesizing machinery.

-__ and __ bind the 30S subunit, preventing premature binding to __.

-__ brings fMet-tRNAf to the mRNA, forming the 30S initiation complex.

-The __ subunit binds, GTP is hydrolyzed by __, the initiation factors leave, resulting in the __ initiation complex. The fMet-tRNAf occupies the P site bound to AUG thereby establishing the reading frame.
- ribosomal P site; 70S
- initiation factors (IF)
-IF1; IF3; 50S
- IF2 (with GTP)
- 50S; IF2; 70S
- elongation factors deliver __ to the ribosome
-__ is one of the most abundant proteins in bacteria
-__ inserted into __ site by __ during chain elongation
-Hydrolysis of GTP causes conformational change in __ – releases __
- aminoacyl tRNA
- EF-Tu
- aa-tRNA; A; EF-Tu
- EF-Tu; aa-tRNA
-Only __ form of EF-Tu can bind aa-tRNA.

-__ helps

-So one __ hydrolyzed for each tRNA brought by EF-Tu
- GTP bound
- EF-Ts; GDP
-The __ is now primed to catalyze peptide bond formation.

-Peptide bond formation, a thermodynamically favorable processs, is catalyzed by a site on the __ RNA, called the __. The 23S RNA is a component of the 50S subunit.

- __ catalyzes peptide bond synthesis
- ribosome
- 23S RNA; peptidyl transferase center
- peptidyl transferase
-Formation of a peptide bond – __ of amino group acts as nucleophile attacking __ of peptidyl-tRNA
- nitrogen; carbonyl carbon
-Formation of the peptide bond is followed by GTP-driven translocation of __ and __
- Elongation factor __ uses the energy of GTP hydrolysis to translocate the __ by one codon.

-Upon translocation, the peptidyl-tRNA is fully in the __, the __ is vacant, and uncharged tRNA is in the __, disengaged from the mRNA.

-The polypeptide chain grows from the __ terminal to the __ terminal.
- tRNAs; mRNA
- G; mRNA
- P site; A site; E site
- amino; carboxyl
-Elongation continues with the growing chain exiting through a channel in the 50S subunit until a s__ appears in the __.

-Stop codons are recognized by __. __ and __ catalyze dissociation of the ribosome, mRNA, and attached tRNA in a reaction facilitated by __.
- stop codon; a site
- release factors (RF); EF-G; ribosome release factor (RRF); gtp hydrolysis
- protein synthesis is energetically expensive, __ are cleaved for each amino acid added to a polypeptide chain
- four phosphoanhydride bonds
Eukaryotic protein initiation is different
1.The ribosomes are larger - __ and __ subunits that form the __ ribosome.

2.Protein synthesis begins with a __ rather than __. A special initiator tRNA called __ is required.

3.The initiator codon is always the first __ from the 5’ end of the mRNA. More protein __ are required.

4. the mRNA is __ because of interactions between proteins that bind the 5' cap and those that bind the poly a tail
5.Elongation and termination are similar except that bacteria have __ release factors while eukaryotes have only __.

6.Eukaryotic protein synthesis occurs in the __ and utilizes large complexes associated with the __.
1. 40S; 60S; 80S
2. methionine; formylmethionine; met-tRNAi
3. aug; initiation factors
4. circular
5. two; one
6. cytoplasm; cytoskeleton
Mutations in eukaryotic initiation factor 2 result in __ disease, characterized by the disappearance of brain nerve cells which are replaced by cerebrospinal fluid.
vanishing white matter disease
-Many antibiotics inhibit __ while leaving __ unaffected.

-___ interferes with the binding of fMet-tRNAf and thereby inhibits protein synthesis initiation in bacteria
- bacterial protein synthesis; eukaryotic protein synthesis
- streptomycin
- some antibiotics inhibit protein synthesis
--__ resembles the 3’ end of an aminoacyl-tRNA, and can enter the A site of a ribosome - The peptidyl-__ formed is bound weakly in the A site and dissociates terminating protein synthesis
-- puromycin; puromycin
Diptheria toxin blocks protein synthesis in eukaryotes by inhibiting __
-Corynebacterium diphtheriae, which causes diphtheria, grows in the upper respiratory tract of an infected individual and produces a toxin that inhibits __.

-The toxin covalently attaches ADP-ribose to an amino acid in elongation factor 2, preventing __ and consequently protein synthesis.

-In unimmunized individuals, infection can be fatal.
- protein synthesis
- elongation
-__ is a small highly toxic protein found in castor beans.

-Ricin has a catalytic activity that cleaves __ from a nucleotide in __ that is crucial for binding elongation factors. Protein synthesis is halted.
- ricin
- adenine; 28s RNA
-In eukaryotes, protein sorting directs proteins to distinct organelles.

-In the secretory pathway, proteins are inserted into the __ membrane co-translationally.

-Protein synthesis in the secretory pathway occurs on ribosomes bound to the __. ER with ribosomes bound is called the __.
- er membrane
- er; rough er (rer)
-The synthesis of proteins bound for the secretory pathway begins on __ that are free in the __.

-First, a portion of the nascent protein that contains a specific signal (__) emerges from the ribosome. The signal peptide is a sequence of 9 to 12 __, often located at the __ region of the primary structure

-The __ binds the signal sequence as it exists the ribosome and directs the complex to the ER. Binding of __ to the ribosome halts protein synthesis.
- ribosomes; cytoplasm
- (the signal peptide); hydrophobic amino acids; n-terminal
- signal recognition particle (srp); srp
-Next, the __, an integral membrane with __, binds to the SRP-ribosome complex.

-The __, a protein-conducting channel, accepts the ribosome from the SRP-SRP receptor complex and __ begins again with the protein now passing through the membrane in the translocon
- srp receptor; GTPase
- translocon; protein synthesis
mRNA use is subject to regulation

-Iron transport and storage is carefully regulated.
-__: an iron storage protein in the cell, has translational regulation from its mRNA. A stem-loop structure in the 5’ UTR (iron response element or IRE) binds the IRE-binding protein (IRE-BP) when iron is absent and prevents translation. When present, iron binds the IRE-BP causing it to dissociate from the IRE, allowing translation occur.
- iron
- ferritin
-__, which binds iron-rich transferrin and facilitates its entry into cells, also several IREs located in the 3’ untranslated region.

-When iron is absent, __ binds the __, allowing the mRNA to be translated.

-When iron is present, it binds to the __, which dissociates from the transferrin-receptor mRNA. Without the __, the receptor mRNA is degraded
- transferrin-receptor
- ire-bp; IREs
-__ leads to mRNA degradation induced by the presence of double-stranded RNA, which may be present during certain viral infections.

-__, a ribonuclease, cleaves double-stranded RNA into small fragments, called __ that are bound by a class of proteins called __ to form the RNA induced silencing complex (RISC).

-The complex locates mRNA complementary to the __ and degrades the mRNA.
- RNA interference (RNAi)
- dicer; small interfering RNA (siRNA); argonaute
- siRNA
-MicroRNAs (miRNAS) – __ long
-Regulate gene expression __
-May regulate as much as __ of mammalian protein-coding genes
-Regulate mRNA __ and/or __
-Some miRNAs promote __ of specific mRNAs
-Some miRNAs promote __ or __ of mRNAs
- 21 nucleotides
- post-transcriptionally
- 60%
- stability; translation
- cleavage
- translational repression; deadenylation
-__ can be used to cleave DNA is specific places.

-These bacterial enzymes recognize cleavage sites of four to eight nucleotides in length that are __, then cleave each strand of the DNA.
- restriction enzymes
- palindromic
-DNA fragments generated by restriction enzyme digestion can be separated by gel electrophoresis in __ or __

-The fragments can be visualized by staining with __.

-Specific fragments can be isolated from the gel.
- agarose; polyacrylamide gels
- ethidium bromide
- __, in which all but one reactive group is blocked, can be sequentially linked to a strand attached to an insoluble support, making DNA artificially!

-The newly synthesized DNA can be tagged with 32P or a fluorescent marker
and used as a __,
or could be used as
a __
- probe; primer
-To identify a specific sequence, the separated fragments are transferred to nitrocellulose paper, a process called __, and then exposed to a radioactively labeled probe complementary to the sequence of interest.

-__ identifies the fragment which binds the probe
- southern blotting
- autoradiography
-__ and __ are key tools for forming recombinant DNA molecules
-Digestion of DNA by __ often generates staggered or sticky ends.

-A __ (a piece of DNA readily taken up and replicated by bacteria) is cleaved by the same restriction enzyme. The ends of the fragment and cleaved vector can anneal, and __
is used to covalently join
them, generating
recombinant DNA
- restriction enzymes; dna ligase
- restriction enzymes
- vector; dna ligase
-__ are small, circular DNA molecules used as vectors for DNA fragments to 20kb
-__ autonomously within a host cell
-Carry genes conferring __, used as marker genes for cells carrying vectors
-Generally the best choice for small inserts
-pBR322 was one of the first plasmid vectors - many of the vectors used today were derived from pBR322
- plasmids
- replicate
- antibiotic resistance
- __: Efficient, commonly used vector for delivering DNA into a bacterial cell - especially important for screening libraries - clone insert into a bacteriophage
-Advantage over plasmid vectors is that __ is more efficient than __ - especially for large DNA fragments
-Disadvantage: DNA must be packaged into phage particles __ - can’t have a line of cells that propagates the molecule
- bacteriophage lambda vectors
- transfection; transformation
- in vitro
-__ can replicate in either prokaryotic or eukaryotic cells
-They can be used to transfer __ between prokaryotes and eukaryotes
-Useful for cloning __ in bacteria (can easily manipulate DNA here), and then expressing the gene products in a eukaryotic cell (many eukaryotic proteins are not properly modified in bacterial cells)
- shuttle vectors
- recombinant dna
- eukaryotic dna
-After a cloning vector and insert DNA have been joined in vitro, __ is introduced into a host cell such as E. coli (transformation)
-Only a small percentage of cells take up the DNA
-__: cells are grown under conditions in which only transformed cells survive
- __: transformed cells are tested for presence of the recombinant dna
- recombinant dna
- selection
- screening
six steps in a recombinant dna experiment
1. Preparation of DNA. __ and __
2. Cleavage of __ at __. Insert DNA can be added at specific points that have been cleaved
3. __ of DNA fragments. Joining of the fragments
4. Introduction of __ into compatible host cells. (Genetic transformation)
5. __ and __ of recombinant DNA in host cells. __ allow insert DNA to be replicated in host cells
6. __ of host cells that contain recombinant DNA of interest. Screening a large number of DNA clones for desired fragment
1. dna vector; target dna
2. dna; particular sequences
3. litigation
4. recombinant dna
5. replication; expression; cloning vectors
6. identification
-__ is a double-stranded DNA molecule generated from an mRNA template by __, a retroviral enzyme.

-mRNA is specifically targeted via its __.
- complementary DNA (cDNA); reverse transcriptase
- poly A tail
-__ can be synthesized from all of the mRNA that a cell contains and inserted into a cloning vector to generate a __.

-Clones containing __ from eukaryotes can be expressed in bacteria.
-__ represent all the mRNAs made in a given cell or tissue – so they vary depending on cell type they were constructed from.
-cDNA libraries from mRNA do not include __ or __ (much less complex than genomic libraries)
- complementary DNA; cDNA library
- cDNA
- cDNA libraries
- introns; flanking sequences
-__ complementary to the desired sequence can be used to screen a cDNA library.

-__ containing a cDNA library are used to infect bacteria growing on a petri plate. Sites of infection, called __, contain identical phages.

-A replica of the petri plate is made on nitrocellulose, the DNA is denatured with NaOH and the __ is used to identify plaques containing the cDNA of interest
- probes
- lambda phage; plaques
- radioactive probe
-Complementary DNA can also be inserted into __, which have powerful promoters for transcription as well as the signals needed for translation

-Replica plates are probed with an __ for the protein of interest.
- expression vectors
- antibody
-A __ is made by fragmenting the entire genome of an organism and inserting the fragments into a vector such as λ phage.

-Bacteria are infected with the λ phage, replicas appear and are then screened using radioactive probes for the gene of interest.

-Genomic libraries do not vary between __, and contain __ and __
- genomic library
- tissues; introns; non-coding dna
-__: plasmids that have been engineered to contain regulatory sequences for transcription and translation (strong promoters, ribosome-binding sites, transcription terminators)
-Eukaryotic genes can be expressed in prokaryotes - get expression, though modifications not the same.
-prokaryotic expression vectors
expression of proteins in eukaryotes
-Prokaryotic cells may be unable to produce functional eukaryotic proteins, __ are not the same.
-Some __ are for eukaryotes - contain eukaryotic origins of replication, marker genes, etc.
-These can be transfected into eukaryotic tissue culture cells and the expressed proteins studied or purified.
-Recombinant viruses (adenovirus, vaccinia virus) can also be employed to express proteins in eukaryotic cells
- post translational modifications
- expression vectors
-__ molecules can also be integrated into the genomes of large multicellular organisms
-Creates __: organisms with stably integrated foreign DNA
- recombinant dna
- transgenic organisms
-DNA can be sequenced by controlled termination of __
-__ uses 2’,3’-dideoxynucleoside triphosphates (ddNTPs) which are incorporated at the 3’ end of a growing chain in place of a dNTP

-Since ddNTPs lack a __
group, subsequent nucleotide
addition cannot take place
-Small amounts of ddNTP’s terminate replication of some chains at each step, leaving a set of fragments of different lengths
-Results can be read on a gel (if radioactive sample used), or on a fluorimeter (if fluorescently labeled dNTPS are used)
- replication
- sanger method
- 3' hydroxyl
-The location to be sequenced is determined by the added __

-Strands of different lengths will result, with the last nucleotide added always the dideoxy analog.

-Separation on a gel that allows detection of one nucleotide differences in size allows you to read the sequence
- primer
-The __ is used for amplifying a small amount of DNA - can get enough to study from a very small sample
-Also can increase the proportion of a particular DNA sequence in a mixed DNA population
-Used is analysis of medical samples: diagnosis of infectious diseases, determination of genetic defects, paternity questions, matching of donor organs
-Also used in study of molecular evolution
- polymerase chain reaction (PCR)
-PCR is capable of __ a single molecule of DNA which can then be visualized in a gel or sequenced.

-PCR is used to detect __ in genes that cause cancer, verify the presence of infectious agents in cells, and determine the guilt or innocence of crime suspects.
- amplifying
- mutations
site directed mutagenesis of cloned dna

-Powerful technique to introduce a desired __ directly into a gene
-__ is synthesized containing mutation and flanking sequences of gene
-From __, DNA replication produces a new copy of the mutated gene
-Important in structure-function studies of genes and their protein products
- mutation
- oligonucleotide
- oligonucleotide primer
-The quantity of individual transcripts in a cell can be determined by __

-__ (made from mRNA) is used in PCR reaction along with a dye that fluoresces when binding duplex DNA. The PCR cycle at which fluorescence's becomes detectable is inversely related to the original number of target templates.
- quantitative PCR (qPCR, real-time PCR)
- cDNA
__ or gene chips allow the determination of the expression pattern of a large number of genes simultaneously. cDNAs are attached to a solid support in a defined pattern to generate the microarray. Fluorescently labeled cDNA is then hybridized to the microarray to reveal the expression level of each gene on the chip.
dna microarrays
steps for PCR:

1. Separate DNA duplex by heating to __

2. Add __ to desired region (must know sequence for primers to anneal to!)

3. Add __ (Taq, Vent) - allow replication for a short period.

-__ strands again; repeat __ and __ steps
1. 95C
2. primers
3. dna polymerase
- heat; annealing; replication
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