-
conservative DNA replication
two strands one old, one new
incorrect mode
-
semiconservative DNA replication
one new strand, one old strand, coiled together
correct mode of replication
-
dispersive DNA replication
mixture of old and new on one strand
incorrect mode
-
DNA replication in prokaryotes
-Meselson- Stahl experiment
-DNA replication is semi-conservative
-each new strand of DNA consist of one old strand and one newly synthesized strand
-
Taylor- Woods-Hughes experiment
demonstrated that DNA replication is semiconservative in eukaryotes
-
DNA replication in prokaryotes
begins at one origin of replication
bidirectional
-
replicon
length of DNA that is replicated following one initiation event at a single origin
-
-
-
DNA polymerase
catalyzes DNA synthesis and requires a DNA template and all four dNTPs
-
Can DNA polymerases I, II, III initiation of chain synthesis?
no
-
does DNA polymerase I, II, III have 5-3' polymerization?
yes
-
does DNA polymerase I,II,III have 3'-5' exonuclease activity?
yes
-
which DNA polymerase has 5-3' exonuclease activity?
polymerase I
-
What does DNA polymerase 1,2,3 need to elongate an existing DNA strand
primer
-
which DNA polymerase initates DNA synthesis?
none
-
exonuclease activity
proof reading, fixes wrongly matched polymers
-
DNA polymerase1 is responsible for:
removing the primer
the synthesis that fills gaps produced during synthesis
-
DNA polymerase 3 if responsible for:
5'-3' polymerization essential in vivo
3'-5' exonuclease activity allows proof reading
10 subunits
not involved in repair
-
1. Unwinding of the helix
- Step one
- DnaA intial steps in unwinding
- DnaB and DnaC further opens and destabilizes the helix
- Energy from hydrolysis of ATP used to supply these proteins
-
Single-stranded binding proteins (SSBPs)
Stabilize the open helix
Step one- unwinding the helix
-
helicases
proteins that open DNA structure
DnaA, DnaB, DnaC
-
2. Reducing increased coiling generated during unwinding
- Step 2
- The unwinding creates super coiling
- DNA gyrase stops super coiling
-
DNA gyrase
- Stops supercoiling
- A DNA topoisomerases
-
3. synthesis of a primer for initiation
- Step 3
- Elongation requires a primer with a 3'-hydroxyl group
- Primase used
-
Primase
- Enzyme
- Synthesizes an RNA primer that provides the free 3' hydroxyl required by DNA polymerase 3
-
4. synthesis of DNA strand
- Step 4
- Replication fork moves, one strand is used for template for continuous DNA synthesis
- Opposite strand undergoes discontinues DNA synthesis
-
Leading strand
- Undergoes continues DNA synthesis
- Enlongated
-
lagging strand
- Undergoes discontinues DNA synthesis
- Synthesized into Okazaki fragments- (small fragments each with an RNA primer
-
5. removal of the RNA primers
- DNA polymerase 1
- Removes the primers on the lagging strand
- Replaces the missing nucleotides (fills gaps)
-
6. joining of the gap-filling DNA to the adjacent strand
- Step 6
- Fragments of lagging strand joined by DNA ligase
-
DNA ligase
fills gaps, binds lagging strands of DNA
-
7.Proof reading
Fix any possible mistakes
-
DNA synthesis at single replication fork involves:
- DNA polymerase 3
- Single-stranded binding proteins
- DNA gyrase
- DNA helicase
- RNA primers
- DNA ligase
-
B- subunit clamp
prevents the core enzyme from falling off the template during DNA synthesis
-
Eukaryotic DNA synthesis
- More complex
- More DNA than prokaryotic cells
- Chromosomes are linear
- DNA complexed with proteins
- Multiple origins of replication=faster
-
autonomously replicating sequences (ARSs)
involved in efficient initiation
-
what major forms of enzymes are involved in initiation and elongation ?
pol alpha and gamma
-
Pol alpha
- Low processivity
- Synthesis of RNA primers during initation on leading and lagging strand
-
processivity
length of DNA synthesized by an enzyme before it dissociated from the template
-
Telomeres
- Found on ends of linear chromosomes
- Long stretches of short repeating sequences
- Preserve the integrity and stability of chromosomes
- Problematic to replication
- Can cause gap in replicated strands
-
Telomerase
- Directs synthesis of the telomere repeat sequence to fill the gap
- Ribonucleoprotein with RNA
- Serves as a template for the synthesis of its DNA complement
-
Homologous recombination
- Genetic exchange at equivalent position along two chromo. with substantial DNA sequence homology
- AKA general, homologous, recombination
- Two chromo. similar sequences
-
Genetic recombination involves?
- Endonuclease nicking
- Strand displacement
- Ligatioin
- Branch migration
- Holliday structure
-
Holliday structure
- Duplex seperation
- Miosis
- Holliday junction
-
recombinant duplexes
sequences similar but mixed
Ab Bb
-
gene conversion
- Consequence of DNA recombination
- Nonreciprocal genetic exchange between two closely linked genes
-
DNA is transcribed into
RNA
-
RNA is translated into
protein
-
The genetic sequence of RNA consists of
A U C G
-
coding strand aka
nontemplate strand
-
non-coding strand aka
template strand
-
triplet codon
- Specifies one amino acid
- Provide 64 codes to specify the 20 amino acids
- Contains start and stop signals
-
Stop codons for DNA
- UGA
- UAA
- UAG
- Do not code for any amino acid
-
-
fameshift mutations
- Insert 1 or 2 nucleotide, protein sequence completely changes
- Insert multiples of 3 it does not completely change
-
triplet binding assay
- Determines specific codon assignments
- Ribosomes bind to a specific codon
-
genetic code is degenerate:
- Many amino acids specified by more than one codon
- Only tryptophan and methionine are encoded by a single codon
-
Wobble hypothesis
3 and 1 position in mRNA and tRNA can be modified
-
different initiation points can create
overlapping genes, 2 different gene sequences
-
RNA serves as the intermediate molecule between
DNA and proteins
-
RNA is synthesized on a DNA template during
transcription
-
in a prokaryote translation and transcription occur
at the same time
-
in an eukaryote transcription and translation occur
- Different times
- Different places
-
RNA polymerase
- Directs synthesis of RNA using DNA template
- No primer required in initiation
- Uses ribonucleotides instead of deoxyribonucleotides
-
RNA transcription in procaryotes requires
- RNA polymerase enzyme holoenzyme
- Promoter sequence on DNA template
- Ribonucleotides
-
Initiation of RNA transcription
- RNA polymerase binds to promoter
- Start sequence TTGACA and TATTAT
-
Elongation in RNA transcription
sigma dissociates and elongation begins with core enzymes
-
termination
- Transcription terminates due to hairpin formation in the RNA
- Some depend on Rho termination factor
-
five steps of transcription in prokaryotes
- 1. RNA polymerase binds to promoter
- 2. short sequence of DNA is unwound
- 3. RNA synthesis begins
- 4. sigma factor dissociates
- 5. RNA elongates
-
difference in eukaryote transcription from prokaryots
- Eukaryotes
- Transcription occurs in nucleus and not coupled translation
- Requires chromatin remodeling
- mRNA processing to produce mature mRNAs
- RNA polymerase (I,II,III)
- Promoter needed
- Transcription factors
- Enhancers and silencer
-
Eukaryotic transcription requires: (1-5)
- 1 promoter
- 2.RNA polymerase
- 3.transcription factors
- 4.enhancer and silencer
- 5. ribonucleotides
-
Promoter in eukaryotic transcription
- TATAA box -35 from start site
- GGCCAATCT box -80from start site
-
RNA polymerase in eukaryotic transcription
- I, produces rRNA
- II, produces mRNA,snRNA
- III, produces 5S rRNA , tRNA
-
transcription factors in eukaryotic transcription
1. general transcription factors
2. Gene-specific transcription factors
-
general transcription factors
required for all RNP II mediated transcription and help RNA polymerase II bind to the promoter and initiate basal level transcription
-
gene-specific transcription factors
influence the efficiency or the rate of RNP II transcription
-
Enhancers and silencers in eukaryotic transcription
- Can be upstream, within or downstream (anywhere)
- Can modulate transcription from a distance
-
initiation complex
- RNA polymerase II + general transcription factors
- 35 subunit at promoter
-
mediator
- Associates and enables either positive or negative regulation of initiation
- 20 subunits after initiation complex
-
introns
- Regions of initial RNA transcript that are not expressed in amino acid sequence
- Removed by splicing and exons joined by mature mRNA
-
spliceosome
spice out Pre-mRNA introns
-
alternative splicing
- Multiple ways to put genes together
- Different exons are included in mRNA
- Increases genetic expression
-
amino acid
- Carboxyl group
- Amino acid
- R (radical) group -refers to specific chemical properties
-
peptide bond
- Forms by dehydration reaction between the carboxyl group of the amino acid and the amino group of another
- Bonds amino acids
-
translation
polymerization of amino acids into polypeptide chains
-
translation requires
- Amino acid
- Messenger RNA (mRNA)
- Ribosomes
- Transfer RNA (tRNA)
-
Prokaryotes have how many monosomes
70S
-
Eukaryotes have how many monosomes?
80S
-
Ribosomes 3 sites:
- E-site: exit site
- P-site: peptidyl site
- A-site: amnioacyl site
-
tRNA
CCA sequence at 3' end is binding site for amino acid
-
aminoacyl tRNA synthetase
actives tRNAs with the appropriate amino acid
-
3 steps of mRNA translation:
- Initiation
- Elongation
- Termination
-
Initiation in prokaryotes requires:
- Small and large ribosomal subunits
- Initiation factors
- Charged initiator tRNA
- Mg
- GTP
-
Initiation codon in prokaryotes
-
Elongation in Prokaryotes
- Requires both ribosomal subunits with mRNA, form P (peptidyl) site and A (aminoacyl) site
- Charged tRNA enter A site
- Peptidyl transterase- catalyzes peptide bond between amino acid on tRNA A site and the peptide chain bound to tRNA P site
- Uncharged tRNA moves to E site
- tRNA bound to peptide chain moves to P site
-
Termination in Prokaryotes is signaled by?
-
what does GTP-dependent release factors do in termination?
take off polypeptide chain from tRNA and release it from translation complex
-
why is translation more complex in Eukaryotes?
- Ribosomes are larger than in bacteria
- Transcription and translation are in different locations at different times
- Ribosomes are with endoplasmic reticulum instead of free floating
- Ribosomes must have initiator sequence in proper order (Kozak sequence)
- Requires more factors for initiation, elongation, and termination than bacteria
-
Kozak sequence
initiator tRNA in proper sequence
-
Difference in initiation Prokaryotes 1:
- Initation begins with methionine-not N-formyl-methionine
- tRNA:tRNAi met
- Initator tRNA bears unformylated methionine
-
difference in initiation in prokaryotes 2:
Contain no Shine-Dalgarno sequence to show ribosomes where to start
-
differences in initiation in prokaryotes 3:
requires more initation factors than eukaryotes
-
difference in initiation in prokaryotes 4:
- Eukaryotes contain 2 release factors
- eRF1
- eRF3
-
Polysomes (polyribosomes)
mRNAs with several ribosomes translating at once
-
Prokaryotic regulation can be controlled under ?
positive and negative conditions
-
regulator elements in prokaryotic gene expression are located?
-
molecules that bind to cis-acting sites in prokaryotic gene regulation
transacting elements
-
lactose metabolism in E.coli is regulated by
- An induced system
- Enzymes responsible for lactose metabolism are induced
- Lactose is the inducer
-
Lac operon
- Three structural genes lacZ, lacY, lacA
- Upstream regulatory region with a promoter and operator
-
permease
- lacY
- enzyme that facilitates the entry of lactose into the bacterial cell
-
transacetylase
- lac A
- Involved in the removal of toxic by-products of lactose digestion from the cell
-
lacI gene regulates transcription of the structural genes by producting
- repressor molecule
- which is allosteric
-
allosteric
- interacts reversibly with another mlc.
- Causes 3-D shape change and change in chemical activity
-
no lactose present
- =repressed
- Repressor binds to operator
- Blocks transcription
- No transcription
- No enzymes
-
lactose present
- = induced
- No binding occurs
- Transcription proceeds
- Transcription > mRNA> translation > enzymes
- Operator-binding region altered when bound to lactose
-
Lac operon mutation Oc
- Active=always on
- nucleotide sequence of operator DNA is altered and will not bind with normal repressor
-
Lac operon mutation I-
- Active=always on
- repressor protein is altered/absent and can not bind to operator region
-
Lac operon mutation Is
- Repressed= not on
- Lactose binding region is altered, repressor always bound to operator
-
Bacteria conjugation
- Genetic info from one bacterium is transferred to another
- F+ cells are donor
- F- are recipients
- Fertility factor (F factor)
-
fertility factor
- F factor
- Can donate DNA during conjugation
-
catabolite-activating protein (CAP)
- Represses expression of lac operon when glucose is present
- Used when there is enough lactose and glucose present
-
Catabolite repression
- repression of the expression of lac operon
- Happens when there is enough lactose and glucose
-
CAP positive control
- Absence of glucose
- Presence of lactose
- CAP-binding site and RNA polymerase bind to promoter
- Max expression=respressor not bound to operator and CAP bind CAP-binding site
- Regulated by activator CAP
-
cAMP is required for
- CAP binding
- levels increase as glucose decreases
-
adenylyl cyclase
- repressed by glucose
- Catalyzes the production of cAMP
- Prevents CAP from binding when glucose is present
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