Final Exam Flashcards
how are Both prokaryotes & eukaryotes affected by antibiotics and toxins?
Ribosomes and their components are frequent targets for antibiotics:
-Antibiotics can block exit channels of ribosome
->Can interfere with normal function of accessory factors
(EF-Tu, EF-G)
->Common antibiotics streptomycin, neomycin, gentamycin, tetracyline, spectinomycin interact with small ribosomal subunit, erythromycin & others with large subunit to induce mistranslation or block charged tRNA binding
-Toxins can chemically modify ribosome components
role of Puromycin in Interfering With Translation?
antibiotic that terminates translation by mimicking a tRNA in the A site
How can you control the amount of a protein in the cell?
1) Usual way is to alter transcriptional rate.
2) Another way is to alter the translational rate/protein synthesis, bypassing need to…
a) transcribe RNA (ATP, GTP!)
b) process mRNA (more ATP)
c) shuttle mRNA to cytoplasm from nucleus (more GTP)
* can respond rapidly to external stimuli->Cells can use pre-made mRNA to rapidly make protein on demand
describe how the primary target of regulators (of bacterial translation) is to interfere with the recognition of the RBS by the 30s subunit (2 ways)
- RNA-binding proteins will bind next to RBS and thus prevent 30S binding of 16s rRNA
- >don’t target mRNA’s RBS directly due to RBS site conservation (this stops 30S subunit from binding all mRNAs!) - RNA molecules can themselves block translation by self-binding (mRNA base pairs with itself to mask RBS), usually in polycistronic mRNA -> prevents later ORF translation until earlier ORFs are translated and “unmask” later ORFs (mRNA no longer self bound)
how can Bacterial ribosome synthesis be regulated at level of translation
Ribosomal Proteins Are Translational Repressors of Their Own Synthesis
-Autorepression of ribosomal protein occurs -> repressors bind their own mRNA near RBS, prevent translation
explain how ribosomal protein synthesis is inhibited only when the regulatory/repressor ribosomal protein is in excess
Available ribosomal proteins find and bind the corresponding available rRNA at high-affinity sites which allows proper folding of rRNA & proteins into ribosome complex (allow proper assembly of ribosome)
-if all the rRNA binding sites are occupied, then ribosomal protein will find and bind a “second choice”binding site on their own mRNA (has lower affinity for this mRNA than for rRNA) and this prevents the translation of the ribosomal protein by inhibiting translation of the open reading frame
how do Eukaryotes block the two initiating events of translation if under stress?
under conditions of reduced nutrients or other cell stresses, it is often useful for Eu cells to reduce translation globally. in these instances, two early steps in Eu translation initiation are targeted for inhibition:
- recognition of the mRNA
- targets the 5’ cap-bindng protein eIF4E which later binds to eIF4G. eIF4E-bindinf proteins compete with eIF4G for binding to eIF4E. In unphosphorylated state, eIF4E-BP bind to eIF4E tightly and inhibit translation. 4E-BP phosphorylation is mediated by protein kinases called mTor. (inhibitors of mTor are affective chemotherapy agents) - Initiator tRNA binding to the 40S subunit
- inhibition mediated by phosphorylation of eIF2. eIF2 bound to GTP is required to deliver initiator tRNA to P site of ribosome. phosphorylation causes reduced levels of eIF4-GTP which is needed to transport tRNA and therefore translation initiation inhibited
ferritin is an Fe2+-binding protein that stores iron and controls iron release and is the major regulator of iron levels in the human body. therefore ferritin must be quickly made to respond to iron levels and so transcriptional control is required. How is Ferritin translation regulated ?
Ferritin transcription is regulated by iron-binding proteins called iron regulatory proteins (IRPs). these proteins are also RNA-binding proteins that recognize a specific hairpin structure formed in ferritin mRNA called iron regulatory element (IRE)
- In cells with little iron, the conc. of iron too low to bind IRPs. with no iron bound to the IRPs, they instead bind to the IRE and inhibit the ability of eIF4A/B to unwind hairpin structure which prevents translation from occurring.
- > when conc. of free iron in the cell is elevated, the IRPs bind iron and the IRPs lose ability to bind to the IRE and therefore can’t inhibit translation.
explain how Translation of Yeast Transcriptional Activator Gcn4 Is Controlled by Short Upstream ORFs and Ternary
Complex Abundance in starvation and non-starvation conditions
Gcn4 controls transcription of many amino acid biosynthesis enzymes (Low amino acids -> more Gcn4 -> more A.A. biosynthesis enzymes -> more A.A.s! OR High A.A. -> less Gcn4 -> less enzymes…)
Nonstarvation conditions: Gcn4 not translated
- Gcn4 mRNA has 4 small ORFs called uORF 1-4 that are upstream coding sequence for Gcn4
- once 40S ribosome recognizes first uORF (uORF1), ribo remains bound to RNA and continues scanning for downstream AUG codons.
- The ternary complex (eIF2-GTP + initiator tRNA) needed for scanning, rebinds to the ribo after uORF1 is translated. Once one of the other (inhibitory) uORFs are translated, the ribo dissociates and falls off and Gcn4 is not translated.
Starvation conditions: Gcn4 is translated
-Low A.A. levels means a lot of uncharged tRNAs with
no A.A.s to carry
-activated eIF2 kinase (Gcn2) phosphorylates eIF2 and slows rate at which eIF2 can bind GTP
->Less eIF2-GTP means that the 40S subunit will slows down ternary complex binding due to rarity, can’t bind mRNA without it -> just continues scanning along and misses start codons after uORF1…
->uORFs #2-4 never made, but Gcn4 start codon is recognized and Gcn4 translation occurs (bc it is likely that the Ternary complex will rebind ribo before it reaches Gcn4 encoded region)
how do Prokaryotes rescue ribosomes on defective and thus dangerous mRNAs?
Prokaryotes rescue ribosomes with by terminating defective mRNAs with special tmRNA: part tRNA, part mRNA
tmRNA = transfer-messenger RNA
what is SSrA?
a 457-nucleotide tmRNA that includes a region at it’s 3’ end that strongly resembles tRNA^Ala
- > SSrA can therefore be charged with alanine and bind EF-Tu-GTP. this complex binds to A site of ribo (tmRNA can only do this due to empty A site-> only then is there room for tmRNA tail) and participates in peptidyl transferase reaction and peptidyl-SSrA translocation results in the release of the broken mRNA and then the SSrA acts like mRNA by entering mRNA binding channel of ribo and encodes 10 codons followed by a stop codon.
- > the protein coded by the incomplete mRNA is fused to the 10-amino acid peptide tag which causes protein to be rapidly degraded (prevent from damaging the cell)
what are the two options eukaryote cells have to degrade mRNAs that are incomplete or have premature stop codon
1) nonsense-mediated mRNA decay (premature stop codon-> frameshift error)
2) “non-stop”-mediated mRNA decay (no stop codon)
describe Nonsense mediated mRNA decay
- uses recruited decapping enzymes
- Relies on exon-marking proteins that are usually displaced by ribosome
1. Exon marking complexes recruit Upf$ proteins
2. Upfs interact with ribosome and activate a decapping enzyme
3. 5′ cap on mRNA is rapidly removed
4. mRNA is degraded by a 5′ -> 3′ exonuclease that attacks uncapped mRNA
describe Non-stop mediated mRNA decay
- removes mRNA and protein from incomplete transcripts
- Ribosome translates polyA tail since there is no stop codon (abnormal!)
- This adds multiple lysines to protein (K=AAA, poly-Lys proteins are unstable, rapidly destroyed by proteases)
- Stalled ribosome is rescued by special eukaryotic release factor 3 (eRF3)-like proteins (Hbs1, Dom34, Ski7 but know them as “eRF3 like H-D-S proteins”)
- Ribosome is released and exonucleases are recruited
- mRNA is degraded by exonucleases
Codons specifying the same amino acid are called ?
synonyms
how does the code seems to minimize effect of any mutation?
- Mutating first position of a codon often gives a similar, if not the same amino acid
- Codons with pyrimidines in 2nd position specify mostly hydrophobic amino acids
- Codons with purines in 2nd position correspond mostly to polar amino acids.
- therefore transitions usually replace one aa with a very similar one.
- Change in the 3rd position rarely results in a different amino acid, even due to transversion
explain Wobble in the anticodon
- base at 5′ end of anticodon is not as spatially confined as the other two..- > allows 5′ end of anticodon to form hydrogen bonds with more than one base located at the 3′ end of a codon
- Early work found tRNAs could bind more than one codon because fifth “base” was found in tRNA anticodons, Inosine (made from deaminated adenine, really a nucleoside)
- Inosine (I) in anticodons can pair with several different bases in codon (can wobble between them)
- Non-canonical Watson-Crick base pairing also occurs (G-U)
- wobble explains why tRNAs don’t exist 1 per codon
Why is the Wobble allowed at the 5′ Anticodon?
3-D structure of tRNA shows stacking interactions between the flat surfaces of 3 anticodon bases and 2 following bases
- These position the first (5′) anticodon base at the end of the stack, thus less restricted in its movements
- The 3′ base appears in the middle of anticodon loop stack, resulting in the restriction of its movements
- The 5’ anticodon wobble base moves free since it is at the end of base pair stack and therefore allows tRNA to recognize a maximum of 3 codons (only when Inosine is at 1st (or 5′) anticodon position)
- adjacent base to the third (3’) anticodon base is always a bulky modified purine residue which further restricts ots movements and explains why wobble is not seen in the first (5’) position of the codon
Three Rules Govern the Genetic Code?
- Codons are read only in a 5′ to 3′ direction (to properly define peptide sequence)
- Codons are nonoverlapping and the message contains no gaps.
- The message is translated in a fixed reading frame, which is set by the initiation codon.
What are the 3 kinds of Point Mutations that alter the Genetic Code?
missence, nonsenese and frameshift mutations
describe:
Missense mutation, Nonsense or stop mutation and a Frameshift mutation
Missense mutation
= an alteration that changes a codon specific for one amino acid to a codon specific for another amino acid (can be silent)
E.g., Sickle cell anemia caused by hemoglobin E -> V mutation
Nonsense or stop mutation
=An alteration causing a peptide chain-termination codon.
Frameshift mutation
= insertions or deletions of one or a small number of base pairs that alter the reading frame
Suppressor mutations?
Suppressor mutations (partly) reverse a mutation by another elsewhere
- > They suppress the change due to mutation at site A by producing an additional new genetic change at site B
- > Suppressor Mutations Can Reside in the Same or a Different Gene (site B is can be in the same or a different gene)
Intragenic suppression vs Intergenic
suppression mutation?
intragenic= occurring within the same gene as original mutation, but at a different site
intergenic = occurring in another gene
Reverse (back) mutations?
change an altered nucleotide sequence back to original sequence
e.g., reverse the harmful mutations by a second genetic change
Intragenic suppression examples?
1) missense mutation - original mutation at site A can be reversed through 1 more missense mutation in same gene
2) frameshift mutation – add/delete base to put ORF back into frame again (Protein reverts almost back to normal)
Intergenic suppression mutation example?
Nonsense Suppression
- Mutant tRNA genes suppress effects of nonsense mutations in protein-coding genes-> Mutated tRNAs can rescue nonsense mutations by introducing an amino acid instead of terminating the chain
- They act by reading a stop codon as if it were a signal for a specific amino acid.
- The act of nonsense suppression is a competition between the suppressor tRNA and the release factor
Genomics revolution has confirmed the universality of the genetic code..…with the exception of ??
mitochondrial DNA, the genetic code is slightly different from the standard code
->mitochondrial tRNAs are unusual in how they decode mitochondrial mRNA
-Only 22 tRNAs found in mammalian mitochondria
because the U in the 5′ wobble position of a tRNA is capable of recognizing all four bases in the 3′ of the codon
(instead of just 3 in universal code…)
->Some bacteria, protozoa, and eukaryotes have non-standard codes too
Gene expression is controlled by regulatory proteins. whats the difference between positive and negative regulators?
- positive regulators, or activators increase transcriptional rate, negative regulators, or repressors decrease transcriptional rate
- both are DNA binding proteins that recognize specific sites at or near the genes they control
- which steps in transcription that are stimulated by activators and inhibited by repressors depends on the promotor and regulators in question
why do most activators and repressors act at the level of transcription initiation? what is a reason for regulating at a later step?
it is the most energy efficient step tp regulate (makes sure no energy in wasted in making mRNA that doesn’t end up getting use/translated)
-also, regulation at first step is easier to do (only a single copy of each gene for haploid genome, so only a single promotor on a single DNA molecule needs to be regulated to control expression of a given gene)
However, also good to regulate at later steps too because it allows more inputs (more signals can modify its expression if a gene is regulated at more than 1 step), it also decreases response time (dont have to transcribe/process mRNA before protein is made)
what is the basal level of transcription ?
RNA Pol binding to “naked” promoter is usually weak in the absence of regulator proteins
- > spontaneously change to open complex
- > start low-level transcription
how are promotor sequences on DNA regulated by regulatory proteins?
Repressors bind and block “operator sites” (prevent RNA Pol from binding to the DNA)
-Some activators bind DNA site and then help RNA Pol bind promoters, i.e., recruitment (an examples of cooperative protein binding to DNA)
how do some activators work by allostery and regulate steps after RNA polymerase binding?
Some promoters require activators to stimulate the transition from closed to open complex
->Activators stimulating this type of promoter function by triggering a conformation change in RNA Pol or DNA itself
Activators can change DNA or RNA Pol conformation to “open” by allostery, give an example of both
- The NtrC (Nitrogen regulatory protein C) activator binds with RNA Pol protein, stimulating a change from a closed
to an open promoter complex - MerR activator binds to merT gene promoter to cause DNA to change conformation -> leading to activation
Cooperative binding between gene regulatory proteins at adjacent sites is common but how come regulatory proteins don’t need to be immediately next to each other for function?
distant DNA sites can be brought closer together to help looping with a DNA-bending protein (i.e., “architectural” proteins)
ex. NtrC activates a promoter “from a distance” since its binding sites are ~150 bp upstream of the target promoter (can be artificially placed 1kb away)
Simple vs complex cooperative binding of regulatory proteins
Simple cooperative binding: activator interacts simultaneously with DNA and RNA polymerase to recruit RNA Pol to the promoter
Complex cooperative binding: Can also have activator (or more than one!) bind and activate RNA Pol that is already bound to promoter
Allostery not only a gene activation mechanism, it is also a way regulators are controlled by their specific signals, how?
A typical bacterial regulator can adopt 2 conformations- one can bind DNA, the other cannot -> dependent on signal molecule binding to it which locks the regulatory protein in one or the other confirmation, therefore determining whether or not it can act.
