DNA Replication
The two strands of DNA have opposite directionality and can therefore be described as ______________.
DNA replication is considered to be ______________ because it is composed of one (old) parent strand and one (new) daughter strand.
Antiparallel; Semi-conservative
Transcription begins at specific DNA sequences called __________ or, more specifically, at a sequence referred to as the ___________.
Promoters; TATA Box
The small ribosomal subunit + initiation factors (complex) bind to the 5' methyl-guanine cap, and are scanning for _______ embedded in ____________ to initiate translation.
From left to right, what are the THREE main components of the Lac Operon, excluding the structural proteins.
1. CAP site = binds CAP protein
2. LacP/lac promotor = binds RNA Pol II
3. LacO/lac operator = binds repressor protein
What are "housekeeping" genes? Provide an example.
Example: proteins involved in cellular respiration
Name the SEVEN main enzymes/proteins that are involved in DNA replication, and describe each function in FIVE words or less.
1. Helicase: Unwinds parental DNA strands
2. Single-stranded Binding Proteins: Prevents DNA strands from re-annealing
3.Topoisomerase: Untangles DNA and removes supercoils
4. Primase: Synthesizes segment of RNA primer
5. DNA Pol I: Replaces primer with DNA nucleotides
6. RNA Pol III: Extends primer with DNA nucleotides
7. DNA Ligase: Covalently bonds okazaki fragments
Processing of eukaryotic mRNA involves ___________ of the primary transcript, sorting out the ________, which contain genetic information, and the ________, which are non-genetic, intervening sequences.
Splicing; Exons; Introns
Within the ribosome, the _____ of the mRNA matches up to the ______ of the tRNA in order to deliver the correct _________ to the building protein. However, there are 61 codons and less than 61 types of tRNA due to flexibility, particularly in the 3rd position, which is referred to as __________.
Codon; Anticodon; Amino Acid; Wobble
What is the Lac i gene, and is it a part of the lac operon? Why or why not?
Lac i gene encodes for the repressor protein. It is not part of the operon - it can be located anywhere on the chromosome - once the repressor protein is made, it will find its way to the operon.
Lac i gene has its own promotor - not really regulated, so it is always expressed, and producing repressor proteins.
Name and describe the TWO types of chromatin structure, as it relates to genes expression.
1. Euchromatin (open chromatin): associated with regions of the genomes that have a lot of genes with high levels of expression.
2. Heterochromatin (closed chromatin): gene poor and not much transcription occurring.
Name the FOUR subunits of DNA Pol III and describe their functions in FIVE words or less.
1. Alpha: 5'-->3' polymerase activity
2. Epsilon: 3'-->5' exonuclease (proofreads/degrades)
3. Beta: Clamps complex to DNA (efficiency)
4. Tau: Tethers two complexes together
There are several General Transcription Factors that are required to initiate RNA Pol II.
Name the two transcription factors that were discussed in detail, and identify the two main functions of each.
1. TFIID (w/ TBP): responsible for recognizing and binding to TATA Box sequence, and induces a bend in the DNA that makes it easier to separate the two strands of DNA.
2. TFIIH: Acts as a helicase to separate the DNA strands, and acts as a kinase, adding phosphate groups to the RNA Pol II C-terminal domain. Phosphorylation = promotor clearance, and binds 2 hitchhiker proteins (capping enzyme & cleavage factors).
Name and describe the THREE specific tRNA binding sites within the ribosome complex.
1. E site (Exit) = each tRNA transits through on its way out of the ribosome.
2. P site (Peptidyl) = tRNA in this position are linked to the polypeptide being assembled.
3. A site (Aminoacyl) = area for "next up" amino acid.
Name the THREE structural genes associated with the Lac Operon, and describe each of their functions.
1. LacZ: Beta-galactosidase = enzyme that catalyzes the rxn that cleaves the bond between the 2 monomers (galactose and glucose), and isomerizes the link between the 2 monomers (lactose<->allolactose).
2. LacY: Lactose permease = membrane transport protein - transports lactose into the cell.
3. LacA: Galactoside transacetylase (dispensable).
What is DNA (cytosine) methylation? Give an example of when a cell would do this.
Gene is taken into a heterochromatic state and made unavailable for transcription.
Generally associated with long term or permanent silencing of a gene.
Serves as a binding site for recruiting proteins that can remodel the chromatin structure.
Ex: Cell type specific genes
Ex: Class of genes that are only expressed in embryonic development
Describe the "End Replication Problem". How do the cells deal with this problem?
* 3' ends of linear chromosomes cannot be replicated (terminal oakazaki fragments aren't synthesized because primer cannot be laid down).
*Newly produced daughter strand is missing some nucleotides in the sequence with every round of replication.
* As the DNA shortens, it loses genetic info, which eventually leads to cellular senescence, which is correlated to the aging process in an organism.
* Temporarily solved with telomerase - an enzyme that extends the 3' end with "nonsense" DNA, called telomeres, but these enzymes are only active during embryonic development so telomeres eventually shorten to a critical level, at which point the cell is no longer able to divide.
During processing of eukaryotic mRNA, a modification occurs at the 5' end.
Explain this process and it's function.
The capping enzyme binds tot he 5' end and adds on a guanine nucleotide - added on in opposite orientation - gets methylated.
This 5' methyl-guanine cap protects the mRNA against degradation (by exonucleases) from the 5' end, and is recognize by initiation factors that lead the ribosomal subunits to bind and begin translation.
What are the FOUR properties of the genetic code?
1. Triplet (codons)
2. Redundant/Degenerate: several codons code for the same amino acid
3. Unambiguous: for any given codon, there is one clear amino acid that it codes for
4. Universal: all organisms (essentially) use the same exact genetic code
In the example of the Lac Operon, how does the cell know that lactose is present?
*Repressor "senses" presence of lactose
*DNA binding of repressor is allosterically regulated by allolactose (isomer of lactose)
* No lactose present: repressor binds to operator, preventing transcription
* Lactose present: allolactose (the "inducer") binds to the repressor, preventing it from binding to the operator, allowing transcription
What is DNA Acetylation? Name the enzymes associated with this process.
Acetylation is associated with less condense chromatin --> transcription activation
Histone modification that confers short term, reversible regulation.
HAT = Histone Acetyltransferase = open
HDAC = Histone Deacetylase = closed
Explain why there is a "leading strand" and a "lagging strand" during DNA replication.
* Both DNA strands are replicated simultaneously, but are running in opposite directions; one going 5'-->3' towards the replication fork, and the other going 3'-->5' away from the replication fork.
* DNA Polymerase is only able to add nucleotides onto newly synthesized strands in the 5'-->3' direction, so as the DNA fork moves, exposing new areas of DNA, the strand oriented 5'-->3' towards the fork (matching the movement of helicase) is synthesized continuously, while the strand oriented 5'-->3' away from the fork must be synthesized discontinuously, in fragments.
Name and describe the modification of eukaryotic mRNA that is linked to termination of transcription.
Once the Poly A Signal Sequence signals the end of a gene being transcribed, cleavage factors release the mRNA from RNA Pol II, and Poly A Polymerase synthesizes a Poly A Tail onto the 3' end of the mRNA. This 3' Poly A Tail provides protection from degradation (from 3' end), and increases efficiency of translation (circularization of mRNA makes it easier for ribosomal subunits to reinitiate translation).
Providing AS MUCH detail as you can, describe the different types of mutations, and their effects, that can occur during translation of a protein.
1. Point mutations (base pair substitutions)
A. Silent: Change in BP does not change the AA so there is no effect on the protein
B. Missense: Change in BP changes the AA, which may or may not effect the structure of the protein
C. Nonsense: Change in BP codes for a stop codon which leads to premature truncation of the protein - can have more/less dramatic effects depending on how soon in translation this occurs
2. Insertions and Deletions of base pairs
A. Frameshift (1-2 BP) causing immediate nonsense
B. Frameshift (1-2 BP) causing extensive missense
C. Indel = 2 BP: extra/missing amino acid - effect depends on importance/location of the AA within the protein
In the example of the Lac Operon, how does the cell know that glucose has run out?
* Catabolite repression by glucose - CAP "senses" transport of glucose into the cell
* CAP (catabolite activator protein) binds to CAP binding site near promotor and interacts with RNA polymerase, and stabilizes it on the promoter
* CAP only activates transcription when cAMP is bound to it (cAMP allosterically activates CAP)
* [cAMP] decreased by glucose transport into the cell because glucose blocks the activity of Adenylyl Cyclase which is needed to produce cAMP
Describe histone modification and name the THREE types of functional groups that can be added/removed. Which functional group is most commonly used?
Responsible for changing conditions, but does not directly alter chromatin - instead recruits chromatin remodeling proteins to reorganize the spacing/coiling/condensation.
Functional groups added/removed from N-terminus tails = phosphorylation, methylation, acetylation (most common)