Translation
Name the 3 types of RNA and their functions.
1) tRNA: (aka transfer RNA) "links nucleic acid information to amino acids; physically brings amino acids to the ribosome"
-intramolecular base pairing (H-bonds)
-one part (anticodon) base pairs w/ codon of mRNA
-one end is attached to a specific amino acid
2) mRNA: (aka messenger RNA) "specifies the sequence of amino acids in a protein"
3) rRNA: (aka ribosomal RNA) "component of ribosomes"
How is prokaryotic gene expression regulated? What are its components?
Operons: "segment of DNA which includes... that is involved in a specific metabolic pathway"
-promoter
-operator
-series of genes with related functions
Name the 4 major components of eukaryotic gene expression and briefly describe the steps that occur in each
1) Transcription: chromatin packaging, transcription factors
-chromatin must be opened up before a gene can be transcribed
2) Post-transcriptional: alternative mRNA splicing and mRNA stability
3) Translation: RNA interference
4) Post-translational: folding, chemical modification, transport, activation, degradation
What is a plasmid? Where are they naturally found?
Plasmid: "small, circular molecules of DNA distinct from the chromosome"
-naturally found in bacteria
-often encode genes for antibiotic resistance
What does it mean for a gene to be constitutively expressed? Regulatory expressed?
1) Constitutive: "some gene products are made CONTINUOUSLY"
2) Regulative: "gene product made ON DEMAND: expression can be INDUCED or REPRESSED"
Describe/draw the structure of a ribosome, including all 3 sites
-E site: "holds a tRNA that will exit"
-P site: "holds the tRNA with the growing polypeptide attached"
-A site: "holds an aminoacyl tRNA"
_____ INDUCES expression of B-galactosidase
_____ INHIBITS expression of B-galactosidase
What is the different between negative and positive regulation?
LACTOSE INDUCES expression of B-galactosidase
GLUCOSE INHIBITS expression of B-galactosidase
-Negative regulation: "when repressor is normally bound to operator, and when it gets taken off transcription starts"
-Positive regulation: "when nothing is bound to the operator but transcription does not occur, and then an activator must bind to the operator turn on transcription"
How does a cell regulate the tightness of chromatin packaging? Why is it important that chromatin is open?
1) post translational modification of histones:
-histone acetylation opens up chromatin
-histone acetyl transferases (HATs) add acetyl groups to positively charged lysine residues (loosens chromatin)
-histone deacetylases (HDACs) REMOVE acetyl groups (condense chromatin)
2) DNA methylation:
-covalent modification of DNA that allows for long-term gene silencing
***must have open chromatin to allow DNA to be accessible for transcription factors + RNA polymerase***
What is needed to create a plasmid in a lab?
1) origin of replication
2) an antibiotic resistance gene (e.g, Amp)
3) a cloning site to insert foreign DNA
-Multiple cloning site = DNA sequence that has multiple different restriction sites (nucleotide sequences) that different restriction enzymes recognize
Describe the 4 major alterations in part of chromosomes
1) Deletion
2) Duplication
3) Inversion
4) Reciprocal translocation
Fill in the blank:
1) _____ are synthesized from their ____ terminus to their ____ terminus
2) ____ is "read" by ribosomes from their __' end to their ___' end
3) A single ____ can be translated ____ time(s) by ribosomes; multiple ribosomes can _________ translate the same ___
4) the _____ produced from translation often must be ______ before it is a functional ______
1) POLYPEPTIDES are synthesized from their AMINO terminus to their CARBOXYL terminus
2) mRNA is "read" by ribosomes from their 5' end to their 3' end
3) A single mRNA can be translated MANY time(s) by ribosomes; multiple ribosomes can SIMULTANEOUSLY translate the same mRNA
4) the POLYPEPTIDE produced from translation often must be MODIFIED before it is a functional PROTEIN
Explain the 2 scenarios about how the trp operon functions
1) In the PRESENCE Of tryptophan (trp):
-transcription is BLOCKED
-trp binds to repressor protein AWAY from active site
-shape of repressor changes & repressor can now bind to operator
-repressor binds to operator, BLOCKING RNA polymerase & preventing it from binding to promoter
-other 5 enzymes NOT translated
*NEGATIVE regulation, bc regulatory repressor protein when bound to operator blocks transcription*
2) in the ABSENCE of trp:
-repressor protein CANNOT bind DNA & transcription occurs
-NO trp to bind to repressor protein
-repressor protein in INACTIVE form & cannot bind to operator
-RNA polymerase binds promoter & starts transcription
-mRNA for trpE, D, C, B, A is TRANSCRIBED
-mRNA is translated into protein (5 enzymes)
What are promoters used for? Where are they found? Which genes use them?
What are enhancers? What are silencers? What do they bind
Promoters:
-found upstream of every gene
-same from one gene to next
-required for transcription
-require open chromatin for assembly of basal transcription complex
-general transcription factors are the SAME for every gene
Enhancers: "unique DNA regulatory sequences that can influence gene expression from a long distance upstream or downstream of the gene they regulate"
-affect what happens at promoter + binding activity of RNA polymerase
-enhancers ACTIVATE transcription of corresponding gene
-every gene has its own set of enhancers; combination of enhancers determines where/when gene is transcribed
Silencers: similar to enhancers, but repress gene expression
**silencers + enhancers bind REGULATORY transcription factors**
What are restriction enzymes? What are they used for?
Restriction enzymes: "endonucleases used by bacteria to prevent uptake of foreign DNA"
*most cut PALINDROMIC sequences!*
-bind to specific parts of DNA sequences and then cleave phosphodiester backbone at that position
-sticky (not equal) vs. blunt (equal) ends
*use of a single restriction enzyme to cut gene on both sides of plasmid can yield TWO DIFFERENT recombinant plasmids*
Name & describe the major mutations. What type of mutation does sickle cell anemia result from?
1) Point mutation: "change in a SINGLE nucleotide"
2)Silent mutation: "nucleotide substitution that does NOT change the amino acid sequence"
-also called synonymous mutation
3) Missense mutation: "nucleotide substitution that CHANGES the amino acid sequence"
-also called a NONsynonymous mutation
-ex: sickle cell anemia (Glu replaced by Val)
4) Nonsense Mutation: "nucleotide substitution that creates a STOP codon"
5) Frameshift mutation: "insertion/deletion of 1 or 2 bases that results in disruption/shifting of reading frame"
-type of point mutation
**most harmful mutation
Describe initiation, elongation, & termination of translation
1) Initiation:
-small subunit of ribosome attaches to 5' UTR of mRNA (ribosome binding site & scans for first AUG) in prokaryotes
-attaches to 5' G cap in eukaryotes & scans 5' UTR for AUG codon
-initiator aminoacyl tRNA binds to start codon
-large subunit of ribosomes binds (initiator tRNA occupies P site)
2) ELONGATION:
-incoming aminoacyl tRNA
-peptide bond formation
-translocation
3) TERMINATION:
-release factor protein binds to stop codon in A site and cleaves polypeptide from tRNA
-new polypeptide is released
-ribosomal subunits and other components dissociate
What 2 proteins does E. Coli need to metabolize lactose? What do these proteins do?
-E. coli only makes B-galactosidase in the PRESENCE of LACTOSE
1) B-galactosidase: "enzyme that breaks down lactose into glucose + galactose"
2) Lactose permease: "protein transporter that transports lactose into the cell"
How do regulatory transcription factors influence transcription? How do RTFs influence difference gene expression?
RTFs recruit co-regulators, co-activators (HATs) if RTF is bound to enhancer; or co-repressors (HDACs) if RTF bound to silencer, leading to chromatin remodeling
**Differential gene expression in cells results from production/activation of DIFFERENT regulatory proteins
What is directional cloning & why is it important?
-allows insert to be ligated properly every time it is inserted into plasmid
-uses 2 different restriction enzymes to cut both the gene AND the plasmid; this gives only ONE POSSIBLE ORIENTATION for the gene, which is good if you want bacterium to express certain gene
Why can enhancers function despite being far away from the promoter?
How can we tell where an enhancer is located in a DNA sequence?
1) Pioneer RTFs bind with a DNA sequence called an enhancer
2) binding of pioneer RTFs recruits HATs to open DNA
-additional RTFs bind to enhancers
-open DNA allows GTFs to bind to promoter of gene + recruit RNA polymerase
3) looping of DNA occurs, allowing RTFs and GTFs to interact effectively "nailing RNA polymerase to promoter" and transcription takes place
*Reporter gene: "gene researchers attach [using recombinant DNA technology] to suspected enhancer of another gene of interest to test for expression in cell culture"
Name all 5 major types of post-translation modification of polypeptides
2) proper folding
3) addition of phosphate, lipid, and/or carbohydrate groups
4) removal of part of polypeptide
5) association w/ other polypeptides or cofactors
True or false: IN a differentiated eukaryotic cell, most genes will NEVER be transcribed?
Why/why not? How is this done?
FALSE!
-DNA methylation, used for long-term gene silencing in addition to histone modifications (HDACs) via covalent modification of DNA
DNA methylation associated w/ CONDENSED chromatin
-each different cell type has a different complement of silenced genes
-DNA methyltransferases (DNMTs): add methyl groups to cytosines
-patterns of methylation are STABLE THROUGH SUCCESSIVE GENERATIONS
*DNA methylation is NECESSARY for proper development!
-can directly impede binding of Tis to DNA or recruit chromatin-modifying proteins, which induce more compact/insative chromatin structure
What is cDNA? How is it made? Why is it important?
cDNA: "DNA that is complementary to the mature mRNA transcript, so it represents the genes sequence WITHOUT introns"
-bacteria carrying plasmids w/ cDNA can make large quantity of human growth hormone + insulin, which can be purified to use to treat patients
-made using reverse transcriptase: "enzyme which uses RNA as a template for DNA synthesis"