What does the strand-directed mismatch repair system do?
Detects distortions in the DNA helix from non-complementary pairs that were missed by the 3`--> 5`exonuclease activity of DNA polymerase
What does the strand-directed mismatch repair system do once it has detected a non-complementary pair?
Distinguishes and removes the mismatched nucleotide from the newly synthesized strand. Lagging strand transiently contains nicks called single strand breaks. These breaks are used to identify the newly synthesized strand of DNA and direct repair system to the correct strand. This requires the leading strand to also have transient nicks. Unknown how this works
What happens as the replication fork moves along the double stranded DNA?
Creates super helical tension, or the winding problem
What would the movement of the replication fork require to prevent tension?
Would require rapid rotation of the entire chromosome ahead of the fork. This isn't possible, would require large amounts of energy.
How is super helical tension relieved?
Through the use of topoisomerases to reversibly break the phosphodiester bond in one or both DNA strands.
- Produces transient single strand breaks in phosphodiester backbone.
- Allows two sections of DNA helix on either side of the break to rotate freely relative to each other.
- Uses phosphodiester bond on opposite strand as swivel point.
- Rotation will occur in direction that relieves the tension.
- Resealing of nick is rapid and doesn't require additional energy.
- Produces transient double strand breaks
- Allows one double helix to pass through another
- Requires ATP hydrolysis
- Series of steps occurs:
- 1) Breaks one double helix reversibly to create a gate.
- 2)Allows the second nearby double helix to pass through the gate.
- 3) Reseals the break and dissociates from the DNA.
- Position where DNA helix is first opened.
- Site where DNA synthesis begins
- Eukaryotes contain many replication origins
- Each origin contains two replication forks
- Replication forks stop moving when they collide with a fork moving in the opposite direction or when they reach the end of the chromosome.
When does DNA synthesis occur in Eukaryotic cells?
- During synthesis phase or S phase.
- Lasts ~ 8 hours in mammalian cells
- At the end of S phase, each chromosome has been replicated to produce 2 complete sets of copies.
What is a telomere?
- Characteristic DNA sequence at the ends of chromosomes
- Contains many tandem repeats of short sequence (Humans GGGTTA repeated ~ 100 times at each telomere
- Counteract the tendency of the chromosome to shorten with each round of replication
What is Telomerase?
- Enzyme that elongates the telomere sequence in DNA.
- Replicates the ends of chromosomes each time the cell divides.
- Linear polymer of nucleotides
- Repeating sugar-phosphate backbone
- Nucleotides are going to be linked together by phosphodiester bonds
- Nucleotides are ribonucleotides
RNA consists of what 4 nitrogenous bases?
Adenine, Cytosine, Guanine, and Uracil
Which nitrogenous bases form base pairs?
- G and C with 3 hydrogen bonds
- A and U with 2 hydrogen bonds
Form of RNA
- Single stranded
- Has structural polarity
- Has ability to fold into complex 3D structures.
How does transcription start?
- Starts with the opening and unwinding of a small portion of DNA double helix
- Nitrogenous bases become exposed
- One of the two strands of DNA is going to act as a template for RNA synthesis.
What is the RNA produced called?
- Elongated one nucleotide at a time in the 5 to 3 prime direction
- Released from the DNA template as a single strand
- Much shorter than DNA molecule
- Enzyme that catalyzes the synthesis of the RNA molecule using the DNA template.
- Catalyze the formation of phosphodiester bonds between ribonucleotides
- Unwinds the DNA helix to expose new regions of the template strand for complementary base pairing
- Synthesis in the 5 to 3 prime direction, read 3 to 5 prime.
- Can start de novo
What type of proofreading does RNA polymerase do?
Modest proofreading, if incorrect nucleotide is added, RNA polymerase can backup in the 3 to 5 prime direction and the active site of RNA polymerase performs an excision reaction.
How many RNA copies can be made from a single gene?
Many, in a short amount of time
When does synthesis of additional RNA molecules start?
Prior to the first RNA being completed.
- Transcribed segment of DNA
- In eukaryotes, typically carries information for just one gene
- Codes for either a single RNA molecule or a single protein (or a group of related proteins if the initial nRNA transcript is spliced in more than one way to produce different proteins).
General transcription factors
Proteins whose assembly at the promoter (nucleotide sequence in the DNA to which RNA polymerase binds to start transcription) is required for the binding and activation of RNA polymerase and initiation of transcription.
Proteins which bind to regulatory sequences in DNA to activate transcription. Help attract the RNA polymerase to start point of transcription.
Protein complex allows transcriptional activators to communicate properly with RNA polymerase and with the general transcription factors.
Chromatin modifying enzymes
Allow greater access to DNA present in chromatin and facilitate the assembly of the transcription initiation machinery onto DNA.
TATA Box (TATA Sequence)
Sequence in the promoter region of many eukaryotic genes to which a general transcription factor binds. This sequence specifies the position at which transcription is initiated. Usually located 25 nucleotides upstream of the transcription site.
Initiation of transcription
- TFIID binds to the TATA Box
- Binding of TFIID causes distortion in DNA of TATA box. Brings the DNA sequences on both sides of the TATA box together.
- TFIIB binds near TFIID
- Rest of the general transcription factors and RNA polymerase assemble at promoter. Forms a complete transcription initiation complex.
- TFIIH contains helicase as one of its subunits and hydrolyzes ATP to unwind the DNA helix at the transcription start site, which exposes the template strand of DNA.
What does RNA polymerase do while remaining at the promoter?
Synthesizes short lengths of RNA.
How does TFIIH start elongation?
- TFIIH phosphorylates RNA polymerase on its C-terminal polypeptide tail (C-terminal domain; CTD).
- Causes a conformational change in RNA polymerase which allows it to move away from the promoter and enter the elongation phase of transcription.
- Most of the general transcription factors are released after this.
RNA polymerase II
- Associated with elongation factors in its elongation phase
- These factors are proteins that decrease the likelihood that RNA polymerase II will dissociate before it reaches the end of a gene.
- Both transcribes DNA into RNA and processes the RNA it produces
5 prime methyl cap
- Added when new RNA strand is 25 nucleotides long.
- Modified Guanine nucleotide.
- Requires 3 enzymes working in succession.
3 enzymes for 5 prime methyl cap
- Guanyl Transferase
- Methyl Transferase
Removes a phosphate group from the 5 prime end of the RNA
Adds a Guanosine monophophate (GMP) in reverse linkage.
Adds methyl group to guanosine monophosphate.
Why is 5 prime methyl cap important?
- Signifies the 5 prime end of eukaryotic mRNA.
- Helps cells distinguish mRNA from other RNA molecules.
- Needed to leave the nucleus.
What are eukaryotic genes broken up into?
Small pieces of coding sequence, with expressed and intervening sequences, exons and introns respectively.
Introns and Exons are transcribed into:
How are Intron sequences removed from Pre-mRNA?
RNA splicing sequence
- Each event removes one intron as a lariat and joins two exons.
- A specific adenine in the intron sequence attacks the 5 prime splice site and cuts sugar phosphate backbone of RNA at this site
- Cut 5 prime end of intron becomes covalently linked to the adenine nucleotide, creating a loop in the RNA molecule
- Released 3 prime -OH end of the exon reacts with the next exon joining the two exons together
- Intron is released
Transcripts of many eukaryotic cells are spliced...
... in more than one way. This produces distinct mRNA which gives rise to variant proteins.
- Same gene product can produce a corresponding set of different proteins trough alternative splicing.
- Makes predicting protein sequences solely from the genome sequence more difficulty
- Large assembly of RNA and protein molecules that performs pre-mRNA splicing in eukaryotes
- Recognizes splicing signals on pre-mRNA molecule, brings two ends of intron together, and provides enzymatic activity for the two reaction steps.
- ATP Hydrolysis is required for assembly and rearrangements of the spliceosome
- Rearrangements of the spliceosome are needed for the creation of the active site of the spliceosome.
End of 3 prime pre-mRNA
- Must be processed in order to end the mRNA.
- Singals are encoded in the genome sequence which specify the end of the mRNA molecule
- Signals are transcribed into RNA by RNA polymerase II
- Signals are recognized by RNA binding proteins and RNA processing enzymes.
Examples of RNA Binding proteins/Processing enzymes
- CstF - Cleavage Stimulation Factor
- CPSF - Cleavage and polyadenylation specificity factor (adds multiple adenines)
- These travel with RNA polymerase on the CTD.
- Get transferred to the 3 prime end processing sequence on the RNA molecules as it emerges from RNA polymerase.
- Additional proteins assemble with Cst and CPSF.
RNA cleaved, What is added, and what adds it?
- Poly-A-polymerase (PAP) adds, one at a time, ~20 adenine nucleotides to the 3 prime end of mRNA produced by the cleavage
- PAP does not require a template.
- As the poly-a tail is synthesized and poly-a-bindng proteins assemble onto the tail
- These binding proteins determine the final length of the tail
What happens after the 3 prime end of the mRNA has been cleaved?
- RNA polymerase II continues to transcribe RNA. This RNA lacks a 5 prime cap and rapidly degrades in the 5 prime to 3 prime direction by an exonuclease that is carried on the CTD.
- RNA degradation eventually causes RNA polymerase II to dissociate from DNA.
What happens to the mRNA when it is complete?
Mature mRNA is exported from the nucleus through the nuclear pore complexes in the cytosol.
What will mature mRNA have?
- 5 prime cap
- Poly a tail
- Has been spliced
What happens to improperly processed mRNA?
- Remains in the nucleus, and is degraded by a nuclear exosome.
- Nuclear exosome is a large protein complex that is rich in 3 prime to 5 prime exonucleases.
What is the most abundant RNA molecule?
- rRNA or Ribosomal RNA.
- Cells contain multiple copies of rRNA genes
What are the 4 types of rRNA in eukaryotes.
Each type is present in one copy per ribosome.
- 18 s, 5.8 s, 28 s, are made by chemically modifying and cleaving a single large precurser 45 s pre-RNA which is transcribed by RNA polymerase 1
- 5s is transcribed by RNA polymerase III, and is synthesized from a different cluster of genes, does not require modification.
What is the site for processing rRNA and assembly of ribosomal subunits?
Small Ribosomal Subunit
18s rRNA + proteins
Large ribosomal subunit
5.8 s, .8 s, 5s rRNA + proteins.
Cell differentiation depends on?
Changes in gene expression rather than any changes in nucleotide sequence of the cell's genome.
Types of gene expression regulation -
- Transcriptional control
- RNA processing control
- RNA transport and localization control
- Translational control
- mRNA degradation
- Protein activity control
Controlling when and how often a given gene is transcribed. Most important mechanism for regulating gene expression
RNA processing control
Controlling the splicing and processing of RNA transcripts.
RNA transport and localization control
Selecting which completed mRNA's are exported from the nucleus to the cytosol and determining where in the cytosol they are located.
Selecting which mRNAs in the cytosol are translated
Selectively destabilize certain messenger mRNA molecules in the cytoplasm
Protein activity cotrol
Selectively activate, inactivate, degrade, or localize specific proteins after they have been made.
What do gene regulatory proteins (transcription factors) do?
Turn specific sets of genes on or off.
Outside of DNA helix is studded with?
DNA sequence information
Gene regulatory proteins recognize?
Information on outside of DNA helix without having to unwind or open up the helix.
The edge of each base pair in the DNA helix is...
- ...exposed at the surface.
- A distinctive pattern of H bond donors, H bond acceptors, and hydrophobic patches are exposed and are recognized by gene regulatory proteins.
- This information is found in the minor and major grooves of DNA.
- The major groove is most important for interactions with gene regulatory proteins.
Eukaryotic gene regulatory proteins control transcription when?
- Bound to DNA.
- The binding site is usually far from the promoter.
DNA sequences that control expression of a gene are often spread over?
Long stretches of DNA.
Gene control region
Term used to describe the entire expanse of DNA involved in regulating transcription. This region includes the promoter and all regulatory sequences to which gene regulatory proteins bind.
Gene activation proteins.
- Attract, position, and modify general transcription factors, mediator ,and RNA polymerase at the promoter so transcription can start.
- Often exhibit transcriptional synergy.
Gene repressor proteins
prevent transcription of an adjacent gene. Many act through more than one mechanism at a given time, ensuring robust and efficient repression.
Types of Gene repression
- Competition for binding site with gene activator protein in the same regulatory sequence of DNA
- Binds to an activation domain of an activator protein to inhibit the activity of the activator.
- Blocks assembly of general transcription factors
- Recruits chromatin remodeling complex which returns the promoter region to the nucleosomal configuration
- Attracts histone deacetylase to promoter. Histone acetylation can stimulate transcription initiation. This reverses the modification.
- Attracts histone methyl tranferase to keep the DNA in nucleosomal form
- mRNA into a protein
- Four nucleotides into 20 amino acids
- Consecutive group of three nucleotides in mRNA.
- RNA is a linear polymer of 4 nucleotides.
- 4 x 4 x 4 = 64 possible combinations of three nucleotides
- Only 20 different amino acids exist
- Genetic code is redundant, and some amino acids are specified by more than one codon.
- Each codon specifies either one amino acid or a stop to the translation process.
- RNA sequence can be translated in any one of three different reading frames, depending on where the decoding process begins.
- Only one of the three possible reading frames in the mRNA encodes the required protein. This determines the proper reading frame to use.
Translation of mRNA depends on??
How long are tRNA's?
Approximately 80 nucleotides.
Four short segments are folded into?
Double helical structures
Two regions of unpaired nucleotides that are crucial to tRNA function?
- First region forms the anticodon set of the three consecutive nucleotides that pairs with complimentary codon in mRNA
- Other region is the 3 prime end of the tRNA. This is the site where the amino acid that matches the codon is attatched to the tRNA
There is more than one tRNA for ?
Many of the amino acids
Some tRNAs can bind to ?
- More than one codon
- tRNAs require accurate base pairing only at the first two positions of the codon and can tolerate a wobble or mismatch at the third position.
Eukaryotic tRNAs are synthesized by?
- RNA polymerase III
- Snythesized as larger precurser tRNAs (pre-tRNAs) which are trimmed to produce mature tRNA.
All tRNA's are...?
Modified chemically. Nearly one in ten nucleotides in each mature tRNA is an altered version of the standard G,U,C,A ribonucleotide. Over 50 types of modifications rae known. Modifications increase codon recognition by the correct transfer tRNA or increase the accuracy of the amino acid attachment to tRNA.
- Enzyme that covalently couples each amino acid to its appropriate set of tRNA molecules
- Most cells have a different Aminoacyl-tRNA synthetase for each amino acid.
- Twenty amino acids means 20 aminoacyl-tRNA synthetases.
Amino acids are attached to the ___ end of tRNA.
3 prime end
What is required for aminoacyl-tRNA synthetase to add the amino acid?
- ATP Hydrolysis
- A high energy bond is formed between the amino acid and tRNA
- The energy of this bond is used during protein synthesis to covalently link the amino acid of the growing polypeptide chain.
Two stop process of selection of amino acid by aminoacyl-tRNA synthetase
- 1. Correct amino acid has the highest affinity for the active site pocket of its aminoacyl-tRNA synthetase. Correct amino acid has been covalently linked to AMP.
- 2. A discrimination step occurs after the amino acid has been covalently linked to AMP. When tRNA bind to aminoacyl-tRNA synthetase, the aminoacyl-tRNA synthetase tries to force the amino acid into a second pocket(editing site) in the aminoacyl-tRNA synthetase. The dimensions of this editing site exclude the correct amino acid but allow access by the other 19 amino acids. Any amino acids that enter the editing site are the incorrect amino acids, and therefore are hydrolyzed from adenosine monophosphate, and are released from the aminoacyl-tRNA synthetase.
The fundamental reaction of protein synthesis is?
The formation of the peptide bond.
Peptide bonds form between?
Carboxyl group at the end of the growing chain, and the free amino group of the incoming amino acid
Growing carboxyl end of polypeptide chain remains?
Activated by ts covalent attachement to tRNA (Amino acid attached to tRNA forms peptidyl tRNA).
Headgrowth type polymerization.
Each amino acid added to the growing peptide chain carries with it the activation energy for the addition of the next amino acid.
- complex catalytic machine
- made from more than 50 different ribosomal proteins and several rRNA molecules.
- Composed of one large and small subunit
Small ribosomal subunit provides?
Framework on which tRNA can be accurately matched to codons on mRNA.
Large ribosomal subunit catalyzes
The formation of peptide bonds
Ribosomes contain ___ RNA binding sites.
- One site binds mRNA.
- Three sites bind tRNA. These sites are the A-site, P-site, and E-site.
Translation elongation Step 1
tRNA carrying the next amino acid binds to the A-site by forming base pairs with the mRNA codon positioned in the A-site. Both the P-site and the A-site contain peptidyl tRNAs. The tRNA in the E-site is released.
Translation elongation Step 2
Carboxyl end of polypeptide chain is released from tRNa at the P-site and is joined to the free amino group of the amino acid linked tRNA at the A-site forming a new peptide bond. The reaction is catalyzed by peptidyl transferase contained in the large ribosomal subunit.
Translational elongation Step 3
Large ribosomal subunit moves relative to mRNA held by the small ribosomal subunit Shifts the cceptor stems of the two tRNAs to the E-site and the P0site of the large ribosomal subunit.
Translational elongation Step 4.
Small ribosomal subunit and its bound mRNA move exactly three nucleotides. Resets ribosome so it is ready to receive the next peptidyl tRNA.
Site where protein synthesis beings sets the ...?
Initiator tRNA always carries?
The amino acid methionine
N-terminus of newly synthesized proteins is?
- Usually removed later by specific protease.
Initiation of translation in Eukaryotes
- Initiator tRNA-methionine complex (met-tRNA) and eukaryotic initiation factors (eIF) are loaded ino the small ribosmal subunit. Met-tRNA binds directly to P-site.
- Small ribosomal subunit binds to the 5 prime end of the mRNA. This is recognized by virtue of its 5 prime methyl cap and 2 bound initiation factors known as eIF4E and eIF4G.
- Small ribosomal subunit moves along the mRNA in 5 to 3 prime searching for the first AUG. Adiitional initiation factors that act as ATP-powered helicases to facilitate the movement of the small ribosomal subunit through the RNA secondary structure.
What happens when the first AUG is encountered?
Initiation factors dissociate. Large ribosomal subunit assembles with the complex completing the ribosome.
End of protein is signaled by?
- One of three stop codons.
- UAA, UAG, or UGA.
Stop codons are not recognized by..?
a tRNA and don't specify an amino acid.
Stop codons signal?
The ribosome to stop tranlation.
Release factors bind to?
Any ribosome with a stop codon positioned at the A-site.
Peptidyl transferase catalyzes the addition of?
- A water molecule instead of an amino acid to the peptidyl tRNA.
- This addition ofwater releases the carboxyl end of the growing polypeptide chain from its attachement to the tRNA molecule in the P-site.
Complete polypeptide chain is released into the?
What happens to the ribosome after polypeptide chain is released?
Ribosome releases mRNA and sperates into small and large subunits.
How many ribosomes can be found on a single mRNA molecule?
Many. Forms a polyribosome or polysome.
Help guide the folding of most proteins.
Molecular chaperones are also called?
Heat shock proteins (HSP's)
Heat shock proteins have a high affinity for ?
Exposed hydrophobic regions on incompletely folded proteins.
ATP-hydrolysis to cycle through a series of binding and releasing proteins throughout the folding process.