Genetics Exam 4

template strand

strand that the RNA is synthesized off of

transcription factor

any protein that binds to dna to facilitate or inhibit gene expression. Includes regulatory proteins like repressors, and activators

coding strand

the strand opposite of the template strand, has identical bases to the synthesized RNA except for T is U

nucleotides in transcription are added to what part of the preexisting RNA?

3’ OH group

does RNA polymerase need a primer?

no

basic requirements of transcription

DNA template, NTPs, proteins

components of an NTP

triphosphate, sugar, base

what is the template, product, and substrate of RNA polymerase?

DNA, ssRNA, NTP

what three components of the DNA template are necessary for transcription

promoter, RNA-coding sequence, terminator

promoter

DNA sequence that is required for transcription, binds RNA polymerase. Has different key elements/consensus sequences depending on eukarytoes vs. prokaryotes

terminator

DNA sequence needed for transcription that signals RNA polymerase to stop transcribing RNA. It is downstream of the gene but does get transcribed.

does the promoter get transcribed

no

sigma factor

a protein that is necessary in bacteria for activating RNA polymerase. It binds to RNA polymerase to form a holoenzyme, which binds the promoter.Without it RNA polymerase cant bind to the promoter

sigma factor turns RNA polymerase from a _____ to a ______

core enzyme (inactive), holoenzyme

-10 and -35 consensus sequence

important parts of bacterial promoter, this is where the holoenzyme binds. Any mutation here slows transcription.

Steps of bacterial transcription

1. sigma factor binds core RNA pol

2. holoenzyme binds promoter

3. DNA unwinds with the help of the holoenzyme

4. NTP is added at the +1 start site and two phosphates are released

5. NTPs keep being added and RNA polymerase moves down the DNA, as it does the sigma factor is released

6. RNA polymerase encounters the terminator, detaches from DNA, and the RNA is released from the DNA. Happens either via rho terminator or independently. (See other flashcards for details)

what bonds are formed between NTPs

phosphodiester

rho dependent termination

in bacteria, A rho protein and a rho utilization site are necessary. This site is upstream of the terminator and rich in C. This is where the rho protein binds and moves toward the 3’ end of the RNA while it is being transcribed. When RNA pol encounters terminator it stops. Rho catches up and when it gets to the DNA/3’ end of the RNA it uses helicase activity to detach the RNA/DNA hybrid

rho independent termination

in bacteria, required DNA to have a section of inverted repeats followed by repeating A!). Once the portion of RNA with the repeats is synthesized, the RNA will base pair at these repeats and form a hairpin structure. This destabilizes the RNA and allows it to separate from the DNA.

inverted repeats

sequences of DNA that are followed downstream by their reverse complement on the same strand. Its like a mirrored sequence expect the two sides not not the same but instead what pairs with the other.

Ex. AGCCCGCC–(nucleotides in the loop part)--GGCGGGCT.

consensus sequence

the most commonly encountered bases at a specific position in a group of related nucleotide sequences. No organism naturally has the perfect consensus sequence, they can only be made in a lab.

• Slashes show two bases being equally common, Y indicates pyrimidines (C, U, T), R indicates purines (R), N indicates that no base is more common.

in eukaryotes, which RNA polymerase makes what kind of RNA?

I: large rRNA

II: pre-mRNA

III: tRNAs, small rRNA

what is the state of chromatin when DNA is transcribed

echromatin/euchromatin/loosely packed

what are the elements of the promoter in eukaryotes

• regulatory promoter: not necessary but supports imitation with activator and regulatory proteins, contains silencers and enhancers

• core: necessary, binds transcription factors and RNA polymerase, located close to +1 site

• -25 tata box: in core promoter, this is where TFIID binds containing tata binding protein.

basal transcription

minimum level of gene expression initiated by just the core promoter, transcription factors, and RNA polymerase

Steps of eukaryotic transcription

1. TFIID containing tata binding protein binds to core promoter, specifically -25 tata box.

2. RNA polymerase and other transcription factors bind

3. DNA undergoes conformational change causing bending and unwinding. RNA pol is also phosphorylated

4. Activator proteins help this process by binding to regulatory sequences

5. RNA polymerase moved along the DNA, adding NTPs. As it moves it leaves transcription factors behind. A wall of amino acids bends DNA at a right angle where RNA is synthesized, RNA exits through a gap in these amino acids.

6. (for RNA pol. II) there is a cleavage site on the mRNA, in which cleavage occurs. the mRNA is transcribed long past the actual protein coding sequence so it cleaved past this. Exonuclease Rat1 enzyme activity then degrades the remaining RNA attached to the DNA.

how are RNA pol. I and III terminated?

I: stopped by proteins called terminator elements that are bound to specific DNA sequences

III: terminated after synthesizing a polyU stretch that causes the RNA to fall off

archaea transcription is more similar to bacteria or eukaryotes

eukaryotes, because similarity in RNA polymerase and TATA box similarity

enzyme

a protein or RNA based catalyst

colinearity, what does it mean for bact. vs. euk.?

means that the order and number of nucleotides in DNA corresponds to the order of amino acids in a protein. In bacteria, it is fully preserved, which means that every RNA that is translated codes for an amino acid. In eukaryotes, colinearity is not continuous. Only exons are collinear and code for amino acids. Introns are spliced out, and even after splicing there are untranslated regions on both the 5’ and 3’ edge bordering the exons.

how does the size of eukaryotic DNA compare to mRNA?

DNA is longer

shine delgarno sequence

in bacteria, 6-10 bases before start codon, binds the ribosome for translation

why is the 3’ UTR important?

has info important for RNA stability and helps regulate translation, also contains poly-A tail which plays a role in translation initiation

why is the 5’ UTR important?

it contains the ribosomal binding site and 5’ cap that is actively involved in translation initiation

mRNA processing

converts pre-mRNA to mature mRNA. mainly a eukaryotic process, involves the addition of a 5’ cap, 3’ tail, and splicing to remove introns

mRNA splicing, why are introns there in the first place?

involves the removal of introns, non-coding DNA in between genes/exons. They are usually there because they have sequences that help regulate gene expression and can serve as an error buffer

what is the mode of splicing in eukaryotic nuclear genes?

with a splice some, and enzyme with five RNAs and 300 proteins

what are the components of a spliceosome and what are they responsible for?

five different RNAs (plus a ton of proteins)

U1: responsible for 5’ cleavage

U2: 3’ cleavage

U4: joining of exons

U5: joining of exons

U6: joining of exons

5’ and 3’ consensus sequence, what is the third important structure that works with these sites

highly conserved sequences at the 5’ and 3’ boundaries of each splice site

The branch point is the third important structure in splicing. It is an A that lies 18-40 bases upstream of the 3’ site and is responsible for lariat formation

steps of mRNA splicing

1. 5’ end is cleaved with U1 and then attached to the branch point to form a lariat

2. 3’ end is then cleaved with U2

3. intron is degraded

4. exons are spliced together with the help of U4, U5, and U6 in the spliceosome

what would happen if the 5’ splice site was deleted?

The intron would be retained. The 3’ site would still be cut but then the RNA would still be all joined back together

what would happen if the 3’ splice site was deleted?

the 3’ end would not be able to be spliced, meaning the lariat forms but the intron is not cut off, and because of the lariat there is no splicing/ exon-exon fusion

what would happen if the branch point was deleted?

no lariat forms, the 3’ end would not be able to be sliced, no exon-exon fusion

5’ capping process

the 5’ end nucleotide’s phosphate is removed, guanine is added via a 5’-5’ bond. This guanine is then methylated

purpose of 5’ cap

increases RNA stability to prevent degredation, helps initiate transcriptiuon via cap binding proteins

why is only mRNA capped?

because the cap is associated with RNA pol II, which produces mRNA.

what is the 3’ tail, processes of its addition

A long (50-25) chain of A’s at the 3’ end of mRNA that gets added during processing.

1. poly-A consensus sequence 11-30 nucleotides from cleavage site that facilitates cleaving

2. 3’ end is cleaved off at cleavage site. The segment cleaved off is rich in U

3. poly-A tail is added to the cleavage site

can only be added to RNA made by RNA polymerase

what does the 3’ tail do

prevents degredation and facilitates ribosomal binding to mRNA, also facilitates splicing

RNA editing, the process

most common in euk, prok dont really do it

occurs when the coding sequence is altered after transcription. Guide RNA base pairs with the mRNA, forming a dsRNA. It pairs based on similarity. Some pairs are complementary, others are not, and that is where the editing happens.The guide RNA will then provide bases for additions or substitutions, and also help with deletions. After editing, the gRNA is removed

guide RNA

a small RNA template that base pairs with transcribed mRNA for editing. It helps add, substitute, or remove bases.

gRNA primer

pre edited RNA

trans-splicing

5’ splice site from one RNA and the 3’ splice site form another molecule to join two exons from different RNA molecules. Basically two exons from different genes are joined. Can alter protein production/make it unpredictable

RNA interference (breifly)

mode of silencing genes, helpful in limiting viral DNA invasion

• two ways: via microRNA and siRNA

microRNA interference

• targets a different gene from which it was transcribed

• does not pair perfectly with the RNA

• inhibits translation

1. naturally made, begins as pri-microRNA. It is a short hairpin structure.

2. Dicer removes the loop of the hairpin, leaving the short dsRNA

3. turned to pre-miRNA by microprocessor complex in nucleus

4. exported to cytoplasm

5. unwound and one short strand forms a RISC complex with proteins

6. this pairs to an mRNA to inhibit translation

siRNA

• targets same gene it is made from

• pairs perfectly with mRNA

• degrades the mRNA

1. dsRNA made synthetically or by viruses

2. cleaved into peices by dicer

3. one short ssRNA segment forms a RISC complex with proteins

4. pairs onyo mRNA and degrades it

auxotrophic mutant

a mutated bacteria that cannot synthesize a nutrient it needs to survive

beedle and Tatum

tested different neurospora mutants on three different media. By observing the different mutants that grew in different mediums, determined the enzymatic pathway for the three different nutrients, and that typically a protein needed for survival is encoded by one gene

degeneracy of code

means that the protein genetic code is redundant, aka more codons are present that what is needed to make 20 AAs. This is a result of some AAs having multiple codons.

synonymous codons

codons that specify the same AA

reading frame

the way in which a nucleotide sequence is read in groups of three for translation, there are three possible reading frames for each mRNA that gets translated. Depending on the reading frame, different AA sequences can be made.

what determines the reading frame

the start codon, AUG

3 termination codons

UAG, UAA, UGA,

is there a tRNA associated with stop codons

no

universal code

almost all forms of life on earth use the same genetic code as we do. However there are some exceptions (most in mitochondrial DNA)

sense codon, how many?

codon that codes for an AA, there are 61

isoaccepting tRNAs

tRNAs that accept the same amino acid but have different anticodons

_____ anticodon(s) recognize(s) _____ codon(s)

one, multiple

how do codons and anticodons pair, where is the wobble position

antiparallel (codon is 5’ →3’ , anticodon is 3’ →5’)

wobble would be 5’ of anticodon, 3’ of codon

wobble position

one anticodon can pair with more than one codon, because the third nucleotide is at what is called a “wobble” position in these synonymous codons. The third nucleotide is often less strict in base pairing than the other two. This means that as long as the first two codons are the same, the anticodon can sometimes pair with multiple codons. As a result, multiple tRNAs can accept the same AA and translation is more efficient.

wobble position rules

5’ end Anticodon G can pair with 3’ end codon U or C

5’ end Anticodon U can pair with 3’ end codon G or A

(gook and uga lol)

aminoacyl tRNA synthetase, steps of its fx

charged tRNA with its specific AA

1. AA reacts with ATP to produce aminoacyl-AMP intermediate and PPi

2. Amino acid is transfered to 3’ end of tRNA, AMP is released

kozak sequence

in euk, a set of nucleotides starting upstream of the start codon and ending at the start codon, facilitates identification of AUG by ribosome

shine delgarno sequence

in prok, 6-8 bases upstream of AUG, is the ribosomal binding site

process of eukaryotic initiation of translation

ribosome binds to the 5’ end of mRNA with the help of cap binding proteins. Ribosome then scans along the mRNA and stops at the Kozak sequence to form the initiation complex with tRNA and initiation factors.

prokaryotic initiation of translation process

there is no Kozak sequence or single start at the start codon. Instead, multiple proteins are transcribed from a single mRNA molecule because prokaryotes are polycistronic. At each open reading frame, there is a ribosomal binding site (SD) which the ribosome can bind to and start elongation.

prok. vs euk. translation

• multiple open reading frames and ribosomal binding sites/one open reading frame and ribosomal binding site per mRNA molecule

• multiple genes have same terminator/one terminator

• multiple proteins from one mRNA (polycistronic)/one protein (monocristronic)

• no 5’ cap/cap binding proteins for ribosomal binding/utilizes cap binding proteins

• shine delgarno binds ribosome, is upstream of AUG/Kozak binds ribosome, is surrounding AUG

IRES

internal ribosome entry sites, they are specialized structures in mRNA. They allow translation to start internally, without the 5’ cap proteins. Occurs in viruses or eukaryotic cells under stress

explain the process of elongation

1. a tRNA is at the P site of ribosome. An incoming tRNA enters the A site, complexed with elongation factors and GTP.

2. GTP turns to GDP, and the amino acid in the P site is transfered to form a peptide bonds with the one on the A tRNA.

3. ribosome translocates/moves along, causing the P tRNA to go to the E site and exit the ribosome. The tRNA in the A site is now in the P site.

4. repeat

what catalyzes peptide bond formation

the ribosome/rRNA

how does translation termination occur

ribosome reaches stop codon (UAA, UAG, UGA). Release factors then cause the polypeptide, tRNA, and mRNA to dissociate from the ribosome.

prokaryotic vs eukaryotic transcription

• one type of RNA polymerase/3 types

• sigma factor/transcription factors

• -10(pribnow) + -35 sequences / -25 sequence (TATA)

• terminates either with rho or by hairpin formation/terminates with cleavage and exonuclease enzyme (for RNA polymerase II) →rho and Rat1 both involve a protein binding to the mRNA and moving along it to its attachment point to the DNA, however one degrades and one does not

• simpler promoter/core (and regulatory) promoter

prokaryotic vs eukaryotic mRNA processing

• continuous colinearity/discontinous

• no introns present so no splicing needed/introns gets spliced

• no 5’ cap or poly-A tail/has 5’ cap and poly-A tail

• editing is uncommon/RNA editing is typical

structural gene

basic gene that encodes a protein

regulatory gene

encodes proteins (like activators or repressors) that control the expression of other structural genes

regulatory element

DNA that is not transcribed but still plays a role in regulating other sequences. Includes operators, enhancers, and silencers (latter two can be found in regulatory promoters!)

negative inducible system

genetic regulation where the default for transcription is OFF. To turn on transcription, the repressor must be unbound from the operator through the binding of an inducer.

negative repressible system

genetic regulation in which the default for transcription is ON. To turn off transcription, a repressor must bind to the operator. This is done through a corepressor binding to the repressor, which then allows it to bind the operator.

inducer

used in negative inducible system, binds repressor so that it unbinds operator

corepressor

used in negative repressible system, binds repressor so that it can bind operator

positive inducible system

default for transcription is OFF, to turn it on an activator must bind to the operator. This is done with a coactivator that binds to activator

coactivator

used in pos inducible system, binds activator so it can bind operator

positive repressible system

genetic regulation in which the default for transcription is ON. To turn off, the activator must be removed from the operator. This is through an inhibitor, which binds the activator to shut it off/release it

inhibitor

used in positive repressible system, binds activator so it unbinds operator

2 components of a holoenzyme, generally

coenzyme and core enzyme

negative control is most common in ____, positive most common in _____

prokaryotes, eukaryotes

is negative repressible anabolic or catabolic

anabolic, it creates a pathway that builds things needed by the body

• therefore need is more constant than only needing to break something down when its in the body

is negative inducible catabolic or anabolic

catabolic, produces proteins that break things down

neg repressible synthesizes uses what kind of regulatory protein

• inactive repressor by default

neg inducible uses what kind of regulatory protein

active repressor by default

pos inducible uses what kind of regulatory protein

inactive activator by default