Term
| Transcriptional regulation involves... (5) |
|
Definition
1. Protein-Protein interactions
2. Protein-DNA interactions
3. Regional and local control of transcription initiation
4. Transcriptional regulators that bind to enhancers
5. Chromatin structure regulation.
PPIRC |
|
|
Term
| True or False: Unlike eukaryotes, transcription and translation occur simultaneously in prokaryotes. |
|
Definition
|
|
Term
| True or false: Transcriptional regulation occur most during the termination stage. |
|
Definition
| False; the initiation stage. |
|
|
Term
| True or false: [mRNA] = [protein]. |
|
Definition
| False due to RNA splicing, editing, and processing. |
|
|
Term
| A gene is a specific DNA segment, which consists of... |
|
Definition
| gene = expressible unit + the sequences controlling expression (e.g. promoter). |
|
|
Term
|
Definition
|
|
Term
| What's the major difference in gene expression between prokaryotes and eukaryotes? |
|
Definition
| Transcription and translation are coupled in prokaryotes and there's less RNA processing. |
|
|
Term
| What DNA sequences comprise the standard prokaryotic gene (expressible unit)? |
|
Definition
1. Transcriptional unit (UTRs and ORF)
2. Coding sequence (ORF)
3. Initiation and termination sequences
4. Regulatory sequences |
|
|
Term
| What's the different between a standard eukaryotic and prokaryotic gene? |
|
Definition
| The presence of introns in the transcriptional unit. |
|
|
Term
| Exons constitute what percentage of the human genome? |
|
Definition
|
|
Term
| The largest fraction of the human genome are made up of? |
|
Definition
| LINE (21%) and SINE (14%). |
|
|
Term
| Regions closer to the ____________ have higher gene density. |
|
Definition
|
|
Term
| What are transposable elements? |
|
Definition
| Movable genes that can insert itself into another part of the genome. |
|
|
Term
| Transposition occurs with ______ frequency but transposable elements are _______ in both prokaryotes and eukaryotes. |
|
Definition
|
|
Term
| In the short term, transposons can influence _____________. In the long term, they can influence _____________. |
|
Definition
| phenotypic expression; genome evolution. |
|
|
Term
| What are the two main types of transposon? |
|
Definition
1. Simple Transposons/Insertion elements (IS elements = contains only the sequences needed for tranposition.
2. Complex/composite transposon = contains 1 or more gene in addition to the sequences needed for transposition |
|
|
Term
| What do simple tranposons/Insertion elements require? |
|
Definition
| a short recognition sequence and short inverted terminal repeats. |
|
|
Term
| What are the major models for transpositon? (packet 6-1) |
|
Definition
1. Direct "cut and paste" transposition
- Two Tn5 transposase monemers bind to end recognition sequences of transposon DNA.
- Tn5 tranposases form a dimer, resulting in a hairpin.
- Tn5 and Hairpin is hydrolyzed and reinserted into another target site in the genome.
2. Direct and Replicative transposition |
|
|
Term
| How can a non-autonomous (Ds) element move itself in the genome? |
|
Definition
| As long as Ds retains its inverted repeat (IR) sequences, it can hitch a ride with an autonomous (Ac) element. |
|
|
Term
| What are some similarities and differences between DNA and replication? |
|
Definition
Similarities:
- Polarity of synthesis (5' to 3')
- Use of DNA template
- Same chemical mechanism involving DNA template, rNTPs, buffer w/ Mg+2, RNA polymerase).
Differences:
- Transcription requires no primer
- Only one template is needed
- Use of RNA polymerase
- RNAP does not have exonuclease (proofreading/repair) activity |
|
|
Term
|
Definition
| transcriptome = all mRNA of a cell at a given time |
|
|
Term
| True or false: Transcriptome can be synthesized de novo. |
|
Definition
| False; there is always some parental RNA present (active or silenced). |
|
|
Term
| Which strand serves as the "coding/sense" strand for mRNA during transcription? |
|
Definition
|
|
Term
| The RNA transcript is nearly identical in sequence to the _____________ stand. |
|
Definition
| non-template (coding/sense) strand |
|
|
Term
| What is a transcription bubble? |
|
Definition
| The point of RNA synthesis where an RNA-DNA hybrid is formed; the bubble travels with RNAP, which results in ssRNA. |
|
|
Term
| What if RNAP followed the helicity of the template DNA? |
|
Definition
| The RNA transcript would wrap around the dsDNA, which would be very difficult to unwind. |
|
|
Term
| How is RNAP able to move in a straight line during transcription? |
|
Definition
DNA turns are "pushed" ahead of the bubble: DNA becomes more tightly wound (positive supercoiling) ahead of the bubble and equivalently unwound behind the bubble (negative supercoiling) so that the linking remains unchanged.
This mechanism is supported using gyrase mutants (cannot relax positive supercoils) and topoisomerases I mutants (cannot relax negative supercoils). |
|
|
Term
| True or false: there are two types of RNA polymerases - DNA dependent (RNAP) and RNA dependent (RdRP). |
|
Definition
|
|
Term
| True or false: Transcription requires a primer. |
|
Definition
| False; no primer is needed, just simple coupling of two nucleoside triphospates. |
|
|
Term
| In which direction does chain elongation during eukaroytic transcription take place? How would you design an experiment to find out? |
|
Definition
From 5' to 3' (nucleotides added to the 3' end).
Since bacterial RNAs have 5'-triphosphate groups, we could use radioactive GTP labeled at the gamma-phosphate to find out. If it's 3' to 5' growth, then it would incorporate the radioactive gamma phosphate. If it's 5' to 3' growth, then it would only incorporate the alpha-phosphate group of the newly added NTP. |
|
|
Term
| Be able to label transcription by RNA polymerase: http://img.photobucket.com/albums/v372/skyriderx2/transcription.jpg |
|
Definition
|
|
Term
| What are the functions of the 2 Mg+2 ions in RNAP during transcription? |
|
Definition
| One Mg ion facilitates the displacement of the pyrophospate while the other Mg ion coordinates the attack by the 3' OH on the alpha-phosphate group of the incoming NTP> |
|
|
Term
| Movement of RNAP along DNA causes ___________ supercoils ahead of the of the transcription bubble and ____________ supercoils behind it. |
|
Definition
| Positive (unwounding); negative (rewinding) |
|
|
Term
| _______________ rapidly eliminate the positive supercoils and regulate the level of negative supercoiling. |
|
Definition
|
|
Term
| True or false: Transcription rates are directly related to the rate at which stable initiation complexes are formed between promoters and the RNAP holoenzyme. |
|
Definition
|
|
Term
| Label the following: http://img.photobucket.com/albums/v372/skyriderx2/transcription2.jpg |
|
Definition
-1 = 1st nucleotide in promoter
+1 = 1st nucleotide in transcript; things before it (upstream) and after it (downstream). |
|
|
Term
| True or false: Prokaryotic upsteam promotor (UP) elements are usually GC-rich and binds to the alpha-C terminus of RNAP. |
|
Definition
| False; prokaryotic UP elements are usually AT-rich whereas in eukaryotes, they're GC rich. |
|
|
Term
| True or false: Mutations in the upstream -35 region and -10 region (TATA/Pribnow box) always increase initiation efficiency and transcription rate. |
|
Definition
| False; they can either decrease (down mutations) or increase (up mutations) transcription rates. |
|
|
Term
| True or false: promoter regions are usually recognized by the alpha subunit of RNAP. |
|
Definition
| False; sigma subunits (there are 7, which determine which genes get transcribed under what conditions). |
|
|
Term
| Describe the steps of the sigma cycle in transcription. |
|
Definition
1. RNAP binds to a sigma subunit, which guides RNAP to a promoter sequence to begin intiation.
2. Once RNA synthesis is initiated, the sigma subunit is replaced by NusA, which begins elongation.
3. Once RNAP reaches a terminator sequence, transcription halts, NusA dissociates from RNAP, and RNAP dissociates from the DNA.
4. RNAP can bind to another sigma subunit, which directs RNAP to its next promoter. |
|
|
Term
| True or false: Transcription is fast and reasonably accurate despite not having any exonuclease (proofreading) activity. |
|
Definition
| True; RNAP is entirely processive. |
|
|
Term
| Why is accuracy in transcription not too much of a problem? |
|
Definition
1. Most genes are repeated transcribed; many copies of a transcript.
2. The genetic code for many residues is degenerate (contains many synonyms).
3. Amino acid substitutions are often tolerated. |
|
|
Term
| true or false: Transcribed RNA is terminated within or just past the AT region. RNA transcripts then formed hairpin structures, stabilized by the GC basepairs. |
|
Definition
|
|
Term
| What are the two types of transcription termination and how are they different? |
|
Definition
1. Rho-independent termination
- Unstable A-U basepairing at the 3' end of the RNA transcript is unstable and causes the RNA to dissociate completely.
2. Rho-dependent termination
- Rho-factor, a hexamer, acts as a helicase to enhance termination efficiency; this requires energy and a specific recognition sequence on the RNA upstream of the termination sequence. |
|
|
Term
| What are the 4 ways RNA can get processed in Eukaryotes? (Hint: SEC-C) |
|
Definition
1. Splicing (removal of introns)
2. Cutting (of RNA).
3. End-modifications (5' cap and 3' polyA tail).
4. Chemical modifications |
|
|
Term
| Why don't we use RNA primers in PCR? |
|
Definition
| They will get hydrolyzed (alkaline hydrolysis). |
|
|
Term
| What does DNA-dependent RNA polymerase do? |
|
Definition
| Transcription: DNA to RNA. |
|
|
Term
| What does RNA-dependent RNA polymerase do? |
|
Definition
| Transcription: RNA to RNA. |
|
|
Term
For the following dsDNA, label the sense and antisense stand, and write out the mRNA transcript:
5'-ATGGGTTGCGTTAAG-3'
3'-TACCCAACGCAATTC-5' |
|
Definition
5'-ATGGGTTGCGTTAAG-3' Sense/coding stand
3'-TACCCAACGCAATTC-5' Antisense/template
mRNA transcript:
5'-AUGGGUUGCGUUAAG-3' |
|
|
Term
| Compare transcription between prokaryotes and eukaryotes. |
|
Definition
Transcription in both requires:
1. Energy
2. Gene to be transcribed
3. Promoter region
4. Terminator (RHO dependent or independent)
5. RNA polymerase
However, eukaryotes have 3 types of RNAP.
DNA differences include genes with introns, enhancers, TSS, termination sites
RNA differences include 5' cap and 3' polyA tail, RNA splicing and editing
Protein differences include chromatin and its modifications, trans factors arrive early. |
|
|
Term
| What are the differences between RNAP I, II, and III in Eukaryotes? |
|
Definition
1. RNAP I = synthesizes precursors of most rRNA
2. RNAP II = synthesizes precursors of mRNA and small RNA.
3. RNA III synthesizes precursors of tRNA, 5S rRNA, other small nuclear and cystolic RNA. |
|
|
Term
| True or false: Mammalian RNAP III has a bipartite promoter: a core promoter element and an upstream promoter element bound by trans factors that recruit RNAP. |
|
Definition
|
|
Term
| What percentage of RNA in a cell is actually mRNA (coding RNA)? |
|
Definition
|
|
Term
| True or false: RNA splicing occurs even when mRNA is still attached to RNA Pol I. |
|
Definition
False; true except it's RNAP II.
- 5' cap added on immediately
- RNA splicing
- PolyA tail added on. |
|
|
Term
mRNA is made by ____________.
pre-rRNA is made by _________.
tRNA is made by _____________.
miRNA and siRNA are made by _________. |
|
Definition
mRNA is made by RNAP II
pre-rRNA is made by RNAP I and III
tRNA is made by RNAP III
miRNA and siRNA are made by RNAP II |
|
|
Term
| What does miRNA and siRNA do? |
|
Definition
miRNA = micro RNA for gene regulation.
siRNA = small interfering RNA for silencing. |
|
|
Term
| What's the definition of a promoter? a core promoter? |
|
Definition
Promoter = all sequences that are important for initiation.
Core promoter = basal promoter ("on" or "off" regualtion) and site of initiation complex assembly. |
|
|
Term
| Describe the general steps in the assembly of transcription initiation complexes. |
|
Definition
1. Binding of TBP (TATA binding protein) and TFII to RNAPII, which binds to promoter region; closed complex.
2. DNA unwinding by TFIIH (helicase) produces opened complex.
3. Phosphorylation of Ser5, then Ser2 on RNAPII's CTD (Carboxy-terminal domain) begins initiation. |
|
|
Term
| What are the 3 major functional groups of trans factors? |
|
Definition
1. General trans factors (GTFs)
- recognize core promoter
- recruits RNAPII
- required for mRNA synthesis
2. Upstream trans factors (UTFs)
- regulates GTFs and RNAPII
- binds to specific recognition sites
- their synthesis is regulated
3. Inducible trans factors (ITFs)
- similar to second group but they are pre-existing and need to be activated.
UTFs and ITFs are regulated in a position- and time-dependent manner. |
|
|
Term
| What's the function of TBP (TATA box Binding Protein)? |
|
Definition
| Cause structural changes in DNA conformation that are important in transcription initiation; kinks DNA by 45 degrees to form a "crank". |
|
|
Term
| TAFs, a subgroup of GTFs, form ________ complexes in transcription initiation sites. |
|
Definition
|
|
Term
| True or false: Activators and RNAPII pre-initiation complex are sufficient in reconstituted systems. |
|
Definition
|
|
Term
| True or false: the RNAPII holoenzyme is formed when the mediator (GTF) binds to the C-terminal domain (CTD) of RNAPII |
|
Definition
|
|
Term
| True or false: Transcription is activated when CTD is phosphorylated at Ser2 and terminated when CTD is phosphorylated at Ser5. |
|
Definition
| False; Transcription is initiated when Ser5 is phosphorylated and is terminated when Ser2 is phosphorylated and Ser5 is dephosphorylated. |
|
|
Term
| True or false: in prokaryotes, transcription and translation occur simultaneously whereas in eukaryotes, translation occur separately in the cytoplasm. |
|
Definition
|
|
Term
|
Definition
|
|
Term
| Which stage of transcription is correlated with 5' capping? 3' polyadenylation? |
|
Definition
|
|
Term
| How does alpha-amanitin in deathcap mushrooms interfere with transcript elongation? |
|
Definition
| It interferes with the NTP bridge/channel in RNAPII |
|
|
Term
| Retroviruses use reverse transcription, meaning ________ is synthesized from _____. |
|
Definition
| DNA synthesized from RNA; RNA to DNA. |
|
|
Term
| What's weird about the retrotransposon lifecycle? |
|
Definition
1. It requires a tRNA primer
2. It uses RNA-dependent DNAP (reverse transcriptase).
3. It uses template switches. |
|
|
Term
| Retroviruses typically have which 3 genes and what is their function? |
|
Definition
1. gag = make up the interior viral core
2. pol = protease, integrase, and reverse transcriptase
3. env = viral envelope proteins |
|
|
Term
| What would happen if the env gene was mutated/lost? |
|
Definition
| Then there would be no viral envelope and the virus would be inactivated; viral particles would drift apart. |
|
|
Term
| Know the general steps of the "retrotransposon lifecycle". |
|
Definition
1. A RNA copy is made via transcription.
2. A tRNA primer is needed to prime the first strand of DNA synthesis.
3. End degradation of RNA copy.
4. First template switch from 5'-LTR to 3'-LTR and completion of first DNA strand.
5. RNAse H degrades most of RNA strand
6. Priming of second strand synthesis
7. Second template switch
8. dsDNA synthesis complete |
|
|
Term
| Describe the steps of retrotransposition integration. |
|
Definition
1. Integrase removes two nucleotides on retroelements
2. integrase cuts host sequence
3. retroelement overhangs are lost
4. Host overhand sequences are duplicated to fill in gaps |
|
|
Term
| What's the purpose of 5' capping of mRNA transcripts? |
|
Definition
| To make the mRNA resistant to degradation by exonucleases; for efficient transport of mRNA outside of the nucleus and to protect against exonuclease degradation. |
|
|
Term
| True or false: 5' capping marks the transition from elongation to termination. |
|
Definition
| False; from initiation to elongation. |
|
|
Term
| When and how does 5' capping occur during transcription? |
|
Definition
| Occurs during early elongation and 7-methylguanosine is added via a 5'-5' triphosphate bridge by a cap-synthesizing complex tethered to the CTD of RNAP II. |
|
|
Term
| True or false: there's no shuffling (no reverse) in RNA splicing. |
|
Definition
| True; splicing needs to be done in order. |
|
|
Term
| Why is splicing important in eukaryotes? |
|
Definition
| Increased recognition of self vs. non-self and increased in complexity of eukaryotes. |
|
|
Term
| Self-splicing introns are examples of ____________. |
|
Definition
|
|
Term
| Describe the steps of self-splicing introns. |
|
Definition
1. The 3'-OH of Guanine attacks the 5' phosphate of the intron.
2. The 3'-OH of the 5' exon attacks the phosphate of the 3' exon, which splices the exon and releases the intron. |
|
|
Term
| What are the 3 major splice junctions? |
|
Definition
1. 5' donor site: G_GUAAGU
2. 3' acceptor site: TAG or CAG
3. branch site: polypyrimidines |
|
|
Term
| How are the splice junctions recognized? |
|
Definition
| 5' splice junction recognized by small nuclear RNA (snRNA). |
|
|
Term
| What structure mediates the splicing of pre-mRNA? |
|
Definition
| spliceosome; splicing can be seen as intron loops with large round particles at the base of the loops (the spliceosomes). |
|
|
Term
| A spliceosome is a combination of... |
|
Definition
| snRNAs, pre-mRNA, and RNA-binding proteins |
|
|
Term
| What's the general functional mechanism of a spliceosome? |
|
Definition
| Association between snRBP coordinate splicing by bring the two splice sites and the branch point into close proximity. |
|
|
Term
| What stage in transcription is polyadenylation a part of? |
|
Definition
|
|
Term
| What are the functions of the 3' polyA tail? |
|
Definition
The Poly(A) tail protects mRNAs from enzymatic degradation and serves as a "counter" of translation cycles.
Eukaryotes tend to protect their mRNA once it's completely processed. |
|
|
Term
| Why is alternative splicing important? |
|
Definition
| It increases genetic variation and eukaryotic complexity; one gene could produce hundreds of different peptides. |
|
|
Term
| rRNA processing in bacteria requires a set of _________________; different from RNA splicing. |
|
Definition
| ribonucleases (III, P, and E). |
|
|
Term
| What's the conserved sequence of the tRNA 3' acceptor stem? |
|
Definition
|
|
Term
|
Definition
| Information transfer from nucleic acid to proteins; different language, different group of chemicals |
|
|
Term
|
Definition
| Information transfer from one nucleic acid to another; same language, same group of chemicals |
|
|
Term
| Explain why the genetic code is a non-overlapping, punctuation-free, degenerate triplet code. |
|
Definition
| How is the code read? Non-overlapping and there's no punctuation after each codon. It's generate because many codons can code for the same amino acid. |
|
|
Term
| How many reading frames can one mRNA strand have? |
|
Definition
|
|
Term
| True or false: The genetic code is an universal code. |
|
Definition
| False; it's a standard code but not universal since mitochondria can have a different code |
|
|
Term
| What's required for translation? |
|
Definition
1. mRNA = messenger for genetic information from DNA to protein.
2. tRNA = physical and informational adapter that carries the proper amino acid to the ribosome; transfer genetic information from mRNA to protein.
3. Ribosome = complicated platform of RNA and protein subunits that coordinates protein synthesis. |
|
|
Term
| If the tRNA anticodon is 3'-UUC-5', then what amino acid is being coded for? |
|
Definition
| mRNA codon will be 5'-AAG-3', which is Lysine (K) |
|
|
Term
| Which position of the mRNA codon is subjected to wobbling? tRNA anticodon? |
|
Definition
| the 3rd nucleotide of the codon or the 1st nucleotide of the anticodon. |
|
|
Term
| True or false: the 3rd nucleotide (3') of an anticodon can wobble. |
|
Definition
|
|
Term
| How can some tRNAs get used for more than one codon? What does this allow? |
|
Definition
Some 5' anticodon base can basepair with more than one nucleotide.
Allows for quick reading. Only at least 32 tRNAs are required instead of 61.
U - A or G
G - C or U
I - U, C, A |
|
|
Term
| What is the adaptor hypothesis? |
|
Definition
| tRNA serves as adaptors that read the nucleic acid code and carry the appropriate amino acid to the ribosome. |
|
|
Term
| What are isoaccepting tRNAs? |
|
Definition
| Different tRNAs that are specific for the same amino acid. |
|
|
Term
| What's the general structure of tRNA? |
|
Definition
Cloverleave structure (can fold into L-shaped structure) consisting of:
- 3'-ACC acceptor stem (highly conserved)
- 5' terminal phosphate
- Anticodon loop
- D-arm
- T-arm
- Variable arm |
|
|
Term
| True or false: There are many non-Watson basepairing in tRNA. |
|
Definition
|
|
Term
| Translation requires not only the choice of the proper amino acid specified by a tRNA but also... |
|
Definition
1. choice of the proper amino acid specified by a tRNA.
2. choice of the proper "charged tRNA" specified by a codon; "second genetic code". |
|
|
Term
| What does the "second genetic code" mean? |
|
Definition
| the choice of the proper "charged tRNA" specified by the codon. |
|
|
Term
| What's the name of the enzyme that link amino acids to tRNA? How many classes of this enzyme are there? |
|
Definition
| Aminoacyl-tRNA synthetase; there are two unrelated classes of aminoacyl-tRNA synthetase, which are specific to different amino acids. |
|
|
Term
| In general, how does aminoacyl-tRNA synthetase link amino acids to tRNA? |
|
Definition
First, the amino acid is activated by ATP to form aminoacyl-AMP and PPi.
The activated aminoacyl is transferred from the enzyme to tRNA to yield aminoacyl-tRNA and AMP. |
|
|
Term
| How do ribosomes select the correct tRNA? |
|
Definition
| Codon-anticodon interactions |
|
|
Term
| How does the correct aminoacyl-tRNA synthetase find the corresponding tRNA? (or how does tRNA find the correct amino acid)? |
|
Definition
| Codon-anticodon interactions and position 73, the discriminator base. |
|
|
Term
| Why is the error rate of aminoacyl-tRNA so low? |
|
Definition
| Because they have proofreading capabilities; they can switch from catalytic (synthetic) mode to editing mode to check for the proper attachment of the correct amino acid to the tRNA. |
|
|
Term
| What are the constituent subunits of a bacterial vs. eukaryotic subunits? |
|
Definition
Bacterial ribosome (70S) = 30S + 50S
Eukaryotic ribosome (80S) = 40S + 60S |
|
|
Term
| What 2 factors contribute to how far something travels in sedimentation? |
|
Definition
|
|
Term
| What are the three parts necessary for translation? |
|
Definition
1. mRNA = conveys genetic information from DNA to protein
2. tRNA = physical and informational adapter; brings proper amino acid to ribosome.
3. Ribosome = provides the platform and machinery for protein synthesis |
|
|
Term
| What are the three ribosomal sites for tRNA? |
|
Definition
1. A = aminoacyl site
2. P = peptidyl site
3. E = exit site |
|
|
Term
| True or false: only 2 tRNAs can be bound to the ribosome at any given time. |
|
Definition
| False; 3 tRNAs can be bound but only 2 can be aminoacyl-tRNAs |
|
|
Term
| Which ribosomal subunit is mostly involved in catalytic activities (peptide elongation)? |
|
Definition
|
|
Term
| Which subunit is involved in the ribosomal recognition processes (mRNA, tRNA binding)? |
|
Definition
|
|
Term
| True or false: even though the ribosome is made up of proteins and RNA, it is the RNA that carries the catalytic activity. |
|
Definition
| True; the ribosome is a ribozyme. |
|
|
Term
| True or false: protein synthesis proceeds from the C-terminus to the N-terminus. |
|
Definition
| False; from the N-terminus to the C-terminus direction. |
|
|
Term
| True or false: ribosomes read mRNA from the 5' to 3' direction. |
|
Definition
|
|
Term
| True or false: active translation occurs on poly-ribosomes (or "polysomes") |
|
Definition
|
|
Term
| Label the following picture of translation: http://img.photobucket.com/albums/v372/skyriderx2/translation.jpghttp://img.photobucket.com/albums/v372/skyriderx2/translation.jpg |
|
Definition
answer from top to bottom:
1. mRNA
2. polypeptides
3. ribosomes |
|
|
Term
| Describe the general steps of the ribosomal peptidyl transferease reaction. |
|
Definition
1. Nucleophilic attack by the amino group of the aa-TNA in the A site results in a new peptide bond.
2. Polypeptide chain is then transferred to the A-site after the nucleophilic attack.
Peptide chain elongation occurs by linkage of the growing peptide (in the P site) to the incoming tRNA's amino acid in the A site = transpeptidation. |
|
|
Term
| What is transpeptidation? |
|
Definition
| When a peptide chain elongates by linking the growing peptide (in the P site) to the amino acid of the incoming aa-tRNA in the A-site. |
|
|
Term
| Describe the general steps of translational initiation in bacteria. |
|
Definition
1. IF-1 helps the dissociation between the 30S and 50S ribosomal subunits (binds to A site)
2. IF-3 prepares the small 30S subunit for reaction
3. mRNA, IF2-GTP, fMet-TRNA, and IF-1 forms preinitiation complex.
4. IF1 and IF3 are released, and 50S subunit joins complex.
5. IF2-GTP hydrolyzed to IF2-GDP.
6. Initation complex is complete and ready for peptide elongation |
|
|
Term
| What is the first residue of bacterial peptides and what does it ensure? |
|
Definition
| N-formyl-methionine; it ensures that the bacterial peptide grows fin the N- to C- terminal direction. |
|
|
Term
| Why does bacteria need a special type of initiator (N-formyl-methionine)? |
|
Definition
| Because bacterial transcripts are polycistronic (one transcript has several cistrons that encode several polypeptides) and each cistron has its own initiator AUG. |
|
|
Term
| What's different about the tRNA^FMet and tRNA^Met? |
|
Definition
They are isoaccepting tRNAs that both accept methionine but tRNA^Fmet has a mismatch pair before position 73.
Formylation of methionine occurs after methionine attaches to adenine on the acceptor stem. |
|
|
Term
| How does a bacterial ribosome determine the site of translation initiation? What specific ribosomal subunit is involved? |
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Definition
| The ribosome binding site recognizes the Shine-Dalgarno sequence on the mRNA (5'-AGGAGGU-3'); the 16S rRNA hybridizes to the Shine-Dalgarno sequence, which helps stabilizes translation initiation. |
|
|
Term
| What's special about the fMet-tRNA binding? |
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Definition
| N-formyl-methionine is the initiator residue that ensures peptide elongation occurs in the N- to C- terminal direction. |
|
|
Term
| What's the function of IF2-GTP in prokaryotic translation? |
|
Definition
| "Adjustment" and proofreading functions. |
|
|
Term
| True or false: Initiation of translation is a key regulatory point in prokaryotes. |
|
Definition
|
|
Term
| What are some major differences between eukaryotic and prokaryotic translation? |
|
Definition
For eukaryotic translation,
- No fMet-tRNA
- No Shine-Dalgarno sequence (no ribosome binding site on mRNA)
- More translation factors
- Scanning is the norm
- Initiation begins at the tranlational start site (TSS), which genberally contains the Kozak consensus sequence |
|
|
Term
| What's the major point of regulation in eukaryotic translation? |
|
Definition
| The binding of Met-tRNA^met to the 40S small subunit, forming the 43S preinitiation complex. |
|
|
Term
| What are the general steps of eukaryotic translation initiation? |
|
Definition
1. Binding of RNA-binding proteins to capped mRNA.
2. Binding of Met-tRNA to the 40S small subunit to form 43S initiation complex.
3. Binding of the 43 initiation complex to mRNA.
4. Binding of eIF5 to form the 48S initiation complex occurs when AUG is recognized.
5. Joining of the 60S large subunit to form 80S initiation complex. |
|
|
Term
| Eukaryotic translation is almost always initiated at the first _____ codon in a good initiation content (Kozak sequence: GCCRCCAUGG) |
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Definition
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|
Term
| mRNAs are bound by ________ complex at the cap. _______ is the "cap-binding protein"). |
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Definition
|
|
Term
| Specifically, the binding of Met-tRNA^Met and _________ to the 40S subunit to form the 43S initiation complex is a major point of eukaryotic translational control. |
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Definition
|
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Term
| True or false: The binding of the 43S initiation complex to mRNA is not mediated by the mRNA itself but by protein-protein interactions between eIF4G and eIF3 bound to the 40s subunit. |
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Definition
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|
Term
| Would would happen if there is not enough eIF2-GTP? |
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Definition
| There would be no binding of the 43S initiation complex to the mRNA and translation would stop. |
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Term
| The ribosome recognizes the first 2 bases of a codon, what would happen if you used an analog? |
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Definition
| You would slow down hydrolysis; more time for proofreading but slow rates of synthesis. |
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|
Term
| What are the 3 major steps in the elongation cycle of bacterial ribosomes. |
|
Definition
1. Decoding: The ribosome selects and binds aa-tRNA whose anticodon matches the codon in the A site.
2. Transpeptidation: transfer of the peptidyl group from P site to the aa-tRNA in the A site; peptide bond formation.
3. Translocation: tRNA in the A site (with the peptide chain) is transferred to the P-site and the [uncharged] tRNA in the P site is transferred to the E-site. |
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Term
| Would would you determine if the catalytic activity of a ribosome is due to RNA or protein? |
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Definition
1. Using ribosomal inhibitors/analogs
2. Using specific ribonucleases or proteases to degrade parts of ribosome
3. Chemical intervention and inhibition |
|
|
Term
| How is translation terminated in bacteria? |
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Definition
| When a stop codon reaches the A site, causing a release factor (RF) to also bind to the A site, causing the peptide chain to be released from the tRNA in the P site. |
|
|
Term
Name the following 3 ribosomal inhibitors (HINT: PCC)
1. An aa-tRNA analog that prevents peptide bond formation and causes premature chain termination.
2. Inhibits peptidyl transferase on the large subunit of prokaryotes.
3. Inhibits peptidyl transferase on the large subunit of eukaryotes. |
|
Definition
1. Puromycin
2. Chloramphenicol
3. Cycloheximide |
|
|
Term
| What's the major difference in translation initiation between prokaryotes and eukaryotes? |
|
Definition
Prokayotes: translation is initiated by recognition and hybridization of the Shine Dalgarno sequence by the 16S rRNA subunit.
Eukaryotes: translation is initiated by protein-protein interactions between the small subunit and cap-binding proteins to recognize AUG within a good context (Kozak consensus). |
|
|
Term
| What are the three stop codons? |
|
Definition
|
|
Term
| What is non-sense mediated decay in yeast? |
|
Definition
| Under normal conditions, the poly A tail stimulates translation when bound to cap-binding proteins but a pre-mature nonsense codon could stop translation. |
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|
Term
| What are the 4 types of post-translational protein modification? |
|
Definition
1. Protein folding - required for activity
2. Protein cutting - by proteases
3. Protein (intein) splicing - removal of inteins
4. Chemical modifications - e.g. histone modifications |
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|
Term
| Before a newly synthesized protein can be active, what must happen first? |
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Definition
| Proteins must be folded into their proper tertiary structure. |
|
|
Term
| What's the difference between protein chaperones and chaperonins? |
|
Definition
Chaperones (e.g. Hsp) = help proteins find the proper structure; this requires ATP and many binding/release events.
Chaperonins = actively fold proteins; forms a multisubunit that fold proteins within its cavity and may unfold improperly folded proteins as well. |
|
|
Term
| True or false: protein chaperones actively fold proteins into their proper structure. |
|
Definition
| False; chaperones help protein find their proper structure but chaperonins do the actual folding. |
|
|
Term
| How does a protein know where to go in the body or cell? |
|
Definition
| proteins have N-terminal signal/targeting sequence that functions as protein zipcodes. |
|
|
Term
| What's the typical structure of a N-terminal signal sequence? |
|
Definition
| Polar residue near N-terminal followed by ~15 hydrophobic residues. |
|
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Term
| True or false: Removal of the N- or C- terminus can activate a protein. |
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Definition
| True; for example, Activation of preproinsulin into insulin requires cleavage of the signal peptide and B chain in addition to disulfide bridge formation. O |
|
|
Term
| Which amino acids can be glycosylated? |
|
Definition
Ser or Thr (O-linked glycosylation)
Asp (N-Linked glycosylation). |
|
|
Term
| What are the two major motifs responsible for protein degradation? |
|
Definition
1. N-end rule = based on differential recognition by specific ubiquitin ligases
2. PEST sequence (sequence rich in Pro, Glu, Ser, Thr) = based on phosphorylation sites that target proteins for degradation |
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|
Term
Know how uniquitin signals for protein degradation (3 main steps and the "ACL" enzymes involved).
http://img.photobucket.com/albums/v372/skyriderx2/ubiquitination.jpg |
|
Definition
1. Ubiquitin-activating enzyme (E1) binds to the Ubiquitin's terminal carboxyl group; requires 1 ATP.
2. Ubiquitin is transferred to one of many ubiquitin-conjugating enzymes (E2), which are process-specific.
3. Ubiquitin ligase (E3) transfer the activated ubiquitin from E2 to the lys amino group of a target protein. |
|
|
Term
| How is ubiquitination specificity achieved? |
|
Definition
| Specificity achieved by the interplay of E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase). |
|
|
Term
| Ubiquitinated proteins are hydrolyzed in the 20S ______________ core particle, which are surrounded by two 19S caps. |
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Definition
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|
Term
| Why is accuracy not too much of a problem for transcription and translation? |
|
Definition
1. Amino acid substitutions are often tolerated.
2. Genes are repeatedly transcribed
3. The genetic code is degenerate |
|
|
Term
| List three major regulatory points in gene expresson. |
|
Definition
1. Transcription initiation
2. Nucleotide availability
3. mRNA processing
4. Translation initiation
5. Amino acid availability
6. Post-translational modifications |
|
|
Term
| True or false: Prokaryotes use translational regulation at the initiation step, not at elongation or termination. |
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Definition
| False; this is true of eukaryotes. Prokaryotes don't use translational control. |
|
|
Term
| What's the difference between positive regulation and negative regulation in transcriptional control? |
|
Definition
Positive regulation = bound activator facilitates transcription.
Negative regulation = bound repressor inhibits transcription. |
|
|
Term
| Reegulation of the lac operon: what happens when both glucose and lactose are present? |
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Definition
| Glucose is the preferred carbon source so lac repressor (LacI) is made constitutively ("always") and binds to the operator: no transcription of Lac operon genes (Lac Z/Y/A). |
|
|
Term
| Reegulation of the lac operon: what happens when only lactose is present? |
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Definition
| Inducer binds to both the free repressor and bound repressor, causes repressor to dissociate from the operator to allow for transcription of Lac operon genes. |
|
|
Term
| True or false: When both glucose and lactose are present, there's still transcription of the Lac operon genes. |
|
Definition
| False; the cell prefers glucose so there's no transcription of Lac operon genes. |
|
|
Term
| True or false: when there's low glucose (high cAMP), cAMP acts as an activator, which causes the lac repressor to dissociate from the promoter and allow for transcription. |
|
Definition
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|
Term
Regulation of the Trp operon: when [Trp] is high, Trp repressor binds to the operator.
Hence, when tryptophan is available, transcription is ___________. |
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Definition
|
|
Term
| True or false: When tryptophan is scare, attenuator (leader peptide) is masked and transcription occurs. |
|
Definition
|
|
Term
| True or false: When [Trp] is low, the ribosome stalls at sequence 1 (the leader peptide), which allows formation of the 3:4 structure that prevents attenuation (termination). |
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Definition
| False; the 2:3 structure prevents attenuation (termination) because sequence 3 can no longer pair with sequence 4; the 3:4 attenuator structure prevents transcription. |
|
|
Term
| True or false: when [Trp] is high, the leader peptide is made faster. |
|
Definition
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|
Term
| Promoters can be complex. What are some examples of different promoter types/elements? |
|
Definition
1. Core promoter
2. Basal promoter
3. Cell-specific module
4. Response module
5. Developmental module |
|
|
Term
| Label the following diagram: http://img.photobucket.com/albums/v372/skyriderx2/promoterQ.jpg |
|
Definition
| http://img.photobucket.com/albums/v372/skyriderx2/promoterA.jpg |
|
|
Term
Define the following:
- activator
- coactivator
- repressor
- corepressor |
|
Definition
- Activator = a factor that increases transcription rate.
- Co-activator = a factor that works with trans factors to increase transcription rate
- Repressor = a factor that decreases transcription rate
- Co-repressor = a factor that works with trans factors to decrease transcription rate |
|
|
Term
| Be able to explain how to achieve activation/repression of transcription. |
|
Definition
Include the following terms:
- Repressor
- Activator
- Inducer
- Mediator
- Promoter |
|
|
Term
| True or false: the binding of different ligands in a ligand-binding domain can cause different activities. |
|
Definition
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|
Term
| When a(n) _____________ is bound to a nuclear receptor, the ligand binding domain (LBD) is positioned so that a coactivator can bind. |
|
Definition
|
|
Term
| When a(n) ____________ binds to a nuclear receptor, coactivator binding to the ligand binding domain (LBD) is blocked. |
|
Definition
|
|
Term
| What's the difference between the action mechanism of a class I and class II nuclear receptor? |
|
Definition
Class I NR (imported into the nucleus) = The presence of a ligand causes the class I NR to be transferred from the cytosol into the nucleus where it binds to a hormone response element (HRE) and acts as a trans factor.
Class II NR (preassembled in the nucleus) = A class II NR is already pre-assembled (bound to DNA) in the nucleus. In the absence of a ligand, it is bound by a co-repressor. In the presence of a ligand, the co-repressor dissociates and a co-activator is recruited. |
|
|
Term
|
Definition
| Heritable states of gene expression without change in DNA sequence. |
|
|
Term
| What is the prototypical epigenetic modification? |
|
Definition
|
|
Term
| In ___________, both cytosine and adenine are methylated whereas in ___________, only cytosine is methylated. |
|
Definition
|
|
Term
| What are the two systems of methylation in eukaryotes? How is specificity determined? |
|
Definition
1. Maintenance methylation
2. "De novo" methylation
specificity is determined by the N-terminus of DNA methyltransferases (DMT) |
|
|
Term
| What are the 5 functions of DNA methylation in eukaryotes? (H-3GX) |
|
Definition
1. Genome defense (methylates foreign DNA)
2. Gene regulation
3. Heterochromatin maintenance
4. X-chromosome inactivation
5. Genomic imprinting |
|
|
Term
| In mammals and plants, DNA methylation usually silences transcription at the ____________ stage. |
|
Definition
|
|
Term
| In mammals and plants, DNA methylation usually occurs to a ____ followed by a G. |
|
Definition
| C; 5'-CpG-3' -> 5'-metCpG-3' |
|
|
Term
In maintenance methylation, DMT recognizes the methyl markers of _________ DNA.
In de novo methylation, it's harder to know where methylation occurs but it usually occurs in _____-rich regions. |
|
Definition
| hemimethylated; GC-rich (CpG regions) |
|
|
Term
| True or false: Loss of DNA methylation in mammals and plants causes development defects, cancer, or death. |
|
Definition
|
|
Term
| True or false: in general, DNA methylation causes gene activation. |
|
Definition
|
|
Term
| True or false: Genes that are insensitive to methylation are usually heavier in gel electrophoresis. |
|
Definition
False; genes that are insensitive to methylation are usually cleaved and lighter.
Genes that are sensitive to methylation are heavier since they are not cleaved. |
|
|
Term
| Which strand is methylated and unmethylated in this picture: http://img.photobucket.com/albums/v372/skyriderx2/methylation.jpg |
|
Definition
Top strand is unmethylated since it's cut.
Bottom strand is methylated. |
|
|
Term
ddm1 mutants show:
- ______ DNA methylation levels
- retrotransposon _____________.
- altered histone modifications
- altered heterochromatin
- defects in _________________. |
|
Definition
| lower; reactivation; genomic imprinting. |
|
|
Term
| True or false: Increase in DNA methylation leads to a decrease in transposon activation. |
|
Definition
|
|
Term
| What did the three DDM1 experiments test for? |
|
Definition
1. change in copy number
- ??
2. change in methylation status
- Mutants have lower methylation levels so they are cleaved more = lots of small bands.
3. expression levels
- RNA loading onto the gel is equal but mutants show more transposon activation and expression than wildtypes. |
|
|
Term
| What is the underlying purpose of genomic imprinting? |
|
Definition
| To determine which genes are inherited from mom or dad. |
|
|
Term
| True or false: CpG islands are found in promoters. |
|
Definition
|
|
Term
| True or false: Under normal conditions, CpG islands are methylated. |
|
Definition
| False; under normal conditions, CpG island are unmethylated and the gene is active. |
|
|
Term
| What kind of genes do you want DNA methylation (gene silencing)? |
|
Definition
| Oncogenes (cancer-causing genes). |
|
|
Term
| What is the methyl donor for all known DNA methyltransferases (DMTs)? |
|
Definition
| SAM (S-Adenosyl-methionine) or AdoMet |
|
|
Term
| Dnmt1 methylates newly synthesized hemimethylated DNA. Hence, it's part of which system of DNA methylation? |
|
Definition
|
|
Term
| Which amino acid residue is critical to the catalysis of DMTs? |
|
Definition
|
|
Term
Active or silent genes?
1. Histone acetylation = ?
2. H3K4 methylation = ?
3. H3K9 methylation = ?
4. Histone methylation = ? |
|
Definition
1. Histone acetylation = active gene
2. H3K4 methylation = active gene
3. H3K9 methylation = silent gene
4. Histone methylation = silent gene |
|
|
Term
| True or false: Histone modifications control DNA methylation. |
|
Definition
|
|
Term
| Which protein is essential for DNA methylation and heterochromatin? |
|
Definition
| Heterochromatin protein 1 (HP1) |
|
|
Term
| Know: Methylation of H3K9 (silenting mark) = recognized by HP1 (adapter protein that binds to several proteins) = interacts with DIM-2 (a DMT), which methylates CpG = methylated CpG maintained by mammals. |
|
Definition
| Methylation of H3K9 (silenting mark) = recognized by HP1 (adapter protein that binds to several proteins) = interacts with DIM-2 (a DMT), which methylates CpG = methylated CpG maintained by mammals. |
|
|
Term
| What triggers DNA methylation in plants? In neurospora? |
|
Definition
| Small RNA. A+T rich regions. |
|
|
Term
| Dicers can cleave longer precursors into either ________ or ________ that can interact with mRNA to cause mRNA degradation or translational inhibition. |
|
Definition
| stRNA (short temporary RNA) or siRNA (small interfering RNA). |
|
|
Term
| What are the 3 ways that siRNA can be produced? [Not on final] |
|
Definition
1. Bidirectional transcription
2. Inverted repeat transcription
3. Aberrant transcription by RdRP |
|
|
Term
| True or false: The hydrolysis of GTP by EF-Tu (EF1) is required for aa-tRNA binding to the ribosomal A site. |
|
Definition
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