Exam 4 - TY Flashcards by Izzie Oberhauser

What direction is DNA synthesis?

5’ - 3’

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What is added in to build a DNA strand? Give 3 examples (building blocks)

Deoxynucleoside Triphosphates (dNTPs)

DATP
dTTP
DGTP

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In order to attach dNTPs, a removal of BLANK BLANK is needed, with only one per nucleoside. These contain energy that is released when broken apart, to attach dNTPs

2 phosphates

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Reply some starting point - BLANK

Origin of replication

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What are some factors of the replisome?

250 BP
Higher conc. Of A and T (easier to pull apart with 2H bonds)
DNA A Proteins (Add tension)
Helicase (DnaB) (Pulls DNA apart)
DNA C (loads helicase)

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How many BP is the replisome?

250BP

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Why is there a higher concentration of A and T at the origin of replication?

Easier to pull apart since they have 2 H bonds compared to 3 of G and C

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Adds tension at the OOR

DNA A proteins

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Identifies the OOR

DNA A proteins

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With DNA proteins, how many can bind?

Up to 40

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How do DNA A proteins add tension?

Put pressure on H-bonds holding DNA strands together

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What is helicase known as?

DNA B

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What helps Helicase load onto strands?

DNA C

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What two things are needed to stabilize single strands of DNA when pulled apart?

Single stranded binding proteins (stabilize single strands)
Topoisomerases (relieve supercooling tension)

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What does DNA polymerase need to build a strand (specific)? And with this, what does it need/look for?

Primer since DNA polymerases cannot start de novo

Requires a free 3’ OH group

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What provides a free 3’ OH group for a primer to attach?

Primase

complimentary, RNA based, 10 bases

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How many bases is a primase?

10 bases

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DNA polymerase has proofreading capabilities. It observes BLANK activity in a BLANK direction, usually BLANK bases per second. It also has different families (BLANK, BLANK, BLANK). Bacteria use family BLANK.

Exonuclease activity

3; - 5’ direction
1000 bases per second
A, B, C
Bacteria: Family C

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What is the main replicative DNA polymerase?

DNA polymerase III

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Bacteria have BLANK DNA Polymerases, with 2 involved in BLANK, and 3 involved in BLANK.

5
2 involved in replication
3 involved in repair mechanisms

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The replication fork is 2 strands, BLANK and BLANK.

Leading (continuous)
Lagging (discontinuous)

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What does the lagging strand contain? How many bases in bacteria compared to humans?

Okazaki fragments

1000 - 2000 bases in bacteria, 100 bases in Euk.

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DNA polymerase has BLANK activity in the BLANK direction. Why can it not grow on an existing chain? How does it get around this?

5’ to 3’ direction
it cannot grow due to the last phosphodiester linkage, which DNA ligaments provides to get around this

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What provides the final phophodiester linkage for DNA Pol I?

DNA ligase

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What is the only DNA polymerase that can do 5’ to 3’? What role does it serve?

DNA Pol. 1 - mainly a backup

As you move away from the OOR, the chromosome folds down away from the other forming a BLANK. It keeps going around circular chromosome until it reaches BLANK at the opposite end? BLANK binds them and blocks against the replication fork.

Theta Structure - Ter sites (opposite OOR) - Tus proteins (bind Ter sites)

BLANK and BLANK unlink interlocked chromosomes, instead of DNA gyrase. Then we see a transfer of chromosomes to BLANK.

Topoisomerase IV and MukBEF - transfer chromosomes to FtsK

E. Coli replicate in as little as 20 minutes, when it should take 40. How?

Replication is a constant process that is always occurring for healthy bacterial cells. Taking chromosome into a cell that is already replicating, since there are multiple levels of replication occurring on the chromosomes taken up by the cells.

DNA REPLICATION # of origins of replication Bacteria: Euk: Archaea:

Bacteria: One Euk: Many Archaea: Few

DNA REPLICATION Direction from origin? Bacteria: Euk: Arch:

Bacteria: Bidirectional Euk: Bidirectional Arch: Bidirectional

DNA REPLICATION Composition of replisome A: DNA Polymerase B: All other components

Bacteria A: Family C B: Unique replisome proteins Euk A: Family B B: conserved replisome proteins Arch A: Family B B: Conserved replisome proteins

DNA REPLICATION Ends of replication in Bacteria, Euk, and Arch

Bacteria: Circular (Ter and Tus proteins) Euk: Linear (Telomerase) Archaea: Circular (we have no idea)

What are plasmids?

Extra chromosomal DNA found in the cytoplasm and not in the supercoiled chromosome

Give some characteristics of plasmids

- Found in cytoplasm - double stranded - circular - bacteria and archaea - nonessential for normal growth - provide an advantage ro antibiotics and virulence plasmids

What advantages to plasmids give?

Antibiotics (resistance genes ‘= R plasmids) Virulence plasmids (help bacteria cause infection by attachment proteins, toxins that damage host tissue, bacterions)

Plasmids are BLANK % of chromosome in size

Some plasmids have higher BLANK, while others have lower

Copy numbers

T/F: Plasmids have a separate replication process, that usually occurs during binary fission.

True

What method of reproduction do plasmids use?

Rolling circle method

Explain the rolling circle method

- Double stranded plasmid - one strand gets nicked - leaves a free 3’ hydroxyl group and a 5’ end exposed - DNA polymerase is able to extend from 3’ OH group - as it works itself around, it displaces the 5’ end - dangles like a tail, completely displaced from plasmid, DNA polymerases then come down here and work on it NO THETA STRUCTURES

What is one huge difference in plasmid vs. chromosome replication?

Plasmids (rolling circle method) do not have theta structures

Transcription =

DNA to RNA

Translation =

RNA to Protein

Transcription is catalyzed by BLANK

RNA polymerase

DNA Vs. RNA

1. Deoxyribose vs ribose 2. Thymine vs. Uracil 3. Double stranded vs. single stranded 4. RNA has intrinsic helicase activity (no separate helicase) 5. RNA can initiate new strands of nucleotides on its own with primers

BLANK has intrinsic helicase activity

RNA

RNA requires ribonucleotide triphosphates in a BLANK direction

5’ to 3’

Does RNA require a primer?

What is the structure of Bacterial RNA polymerase?

(Alpha2, Beta, Beta’, W) + Sigma

Initiation of transcription begins with what?

Finding the promoter

Bacterial Pribnow box

TATAAT

Bacterial Nucleotides:

TTGACA

Eukaryotic Nucleotides:

TATA

BLANK are regions of DNA where RNA transcriptase binds to begin

Promoters

What is the Eukaryotic promoter and what element does it have?

TATA box, with a B-recognition element

What do Euk use to locate promoters? And what does this do?

Transcription factors locate promoters. This turns on genes by binding the promoter regions)

T/F: Either strands can be the template strand, but transcription only occurs in 3’ to 5’

False: 5’ to 3’

Does the promoter transcribe the entire chromosome?

No, only specific genes

What are the two sites in bacteria that transcription can occur at?

Finding the promoter: Pribinow box: TATAAT 35 bases upstream of start codon: TTGACA

In Bacteria, BLANK factors recognize promoters to begin transcription. These are homologous to BLANK in eukaryotes

Sigma factors Homologous to transcription factors

Elongation of transcript can occur de nova, meaning no BLANK is needed. The BLANK factor cues it in, BLANK is added, which means BLANK is no longer necessary.

- Primase - Sigma factor - RNA polymerase - Sigma Factor

What is the primary role of sigma factors? Can more than one be expressed?

Primary role is to get the promoter recognized, then it is removed from the RNA complex once it gets going. Multiple can be expressed, but typically have one main one for transcription

What dictates the termination of transcription?

The DNA sequence, which contains the promoter

What are the two methods of Termination

1. Intrinsic termination method 2. Extra i sic termination method

Intrinsic termination method has everything “within”, so we don’t need extrinsic factors. Looking for high BLANK area with a BLANK. The complimentary bases in the RNA strand fold back together, creating a BLANK. This crowds BLANK and stalls is = dissassociation. Past this, we see a string of BLANK, which pair with U’s , having the weakest interaction following the stem loop structure.

- GC content - inverted repeat - Stem loop structure - RNA polymerase - Adenines

Extrinsic termination method uses BLANK. This binds to the RNA message being made, not the DNA or RNA polymerase. BLANK binds to the BLANK site, within the RNA transcribed. Slides down RNA message to become a termination sequence at the end of the gene, usually involved in stem loop structure. BLANK interacts with the stalled RNA polymerase and allows it to detach.

- Rho proteins - Rho-protein binds to the Rut site - Rho

COMPARING TRANSCRIPTION PROCESS How many RNA polymerases?

Bacteria: ONE EUK: THREE RNA Pol 1 = tRNA and rRNA RNA Pol 2 = mRNA RNA Pol 3 = tRNA and rRNA Arch: ONE

COMPARING TRANSCRIPTION PROCESS Composition of RNA polymerase (subunits)

Bacteria: 4 - 5 subunits Euk: 12 subunits Arch: 11 - 13 subunits

Is archaea RNA pol composition closer to Euk or bacteria ?

Eukaryotes

COMPARING TRANSCRIPTION PROCESS Recognition of promoter

Bacteria: Sigma factor Euk: Transcription factors (multiple) Archaea: Transcription factors (less)

COMPARING TRANSCRIPTION PROCESS Termination of transcription

Bacteria: 1. Intrinsic method 2. Rho-protein mediated Euk: Termination signal w/ terminator protein (not the same as Rho) Archaea: 1. Inverted repeats followed by AT rich DNA sequence 2. Lack inverted repeats but contain repeated runs of thymine’s 3. ETA-termination protein (similar to Rho)

More than one gene in a prokaryotic mRNA transcript =

Polycistronic mRNA

One gene per transcript =

Monocistronic (Euk)

Where do post-transcriptional modification take place for monocistronic transcripts

In the nucleus

What are some post-transcriptional modification we observe ?

1. 5’ cap (7-methylguanosine) = helps initiate translation 2. 3’ End (Poly A tail) = stability, gauges life time of mRNA, 200 nucleotides long, degrades over time

Transcription yield mRNA, and what else?

tRNA = transfer RNA .rRNA = ribosomal RNA

Translation =

Making a protein

What is the language of mRNA?

The genetic code

The genetic code is read is series of triplets called BLANK

Codons

Each codon encodes for a specific BLANK

Amino acid

tRNA is more stable than mRNA, why?

Secondary structure of tRNA prevents ribonuclease action

BLANK, BLANK, and BLANK are required to synthesize proteins which is needed for everything else

MRNA, tRNA, rRNA

What provides longevity ?

Poly A tail

What is involved in the initiation of translation?

5’ cap (7-methylguanozine)

What is the start codon

AUG

What are the stop codons?

UAA UAG UGA

The first two codons are most important for determining the amino acid, the third is not, which is referred to as BLANK

Wobble site

AUG codes for: Euk = Bac =

Euk = Met Bacteria = Formyl methionine

Do stop codons code for amino acids?

What does tRNA do?

Brings amino acids to the ribosome to be incorporated into the growing polypeptide

How long is tRNA?

73 - 93 nucleotides long

tRNA has regions of secondary structure due to intrastate complementarity. What does this do / create?

Allows for greater stability (longevity) = Clover leaf structure

TRNA has regions complementary to the appropriate codon for the amino acid it binds, this includes the BLANK loop and BLANK end

- Anticodon loop - Acceptor end

What are some modified bases in tRNA that add stability?

- Pseudouridine - Dihydrosuridine - Dimethylguanozine

BLANK binds to the appropriate codon for the amino acid bound at the acceptor end (3’ end)

Anticodon

Why is tRNA more stable than mRNA?

TRNA has a secondary clover-leaf structure due to intrastrand base pairing, as well as modified bases (pseudouridine)

IF =

Initiation factor

BLANK, BLANK, BLANK nucleotides at the BLANK end of tRNA forms the acceptor end adding enzyme. This post-transcriptional modification is done by BLANK

C, C, A 3’ end Aminoacyl-tRNA synthetases

What percent of rRNA and protein are in ribosomes at the site of protein synthesis?

60% rRNA 40% protein

Bacteria / Archaea Total = A Small subunit = B = C. Large subunit = D = E and F

Total = 70s Small subunit = 30s —> 16s rRNA Large subunit = 50s —> 5s and 23s rRNA

Eukarya Ribosome Total= Small = Large =

Total = 80s Small = 40s —> 18s rRNA Large = 60s —> 5s, 5.8s, 28s rRNA

The initiation of translation brings together what 3 things?

1. MRNA transcript 2. TRNA bearing the first amino acid of the protein 3. Two ribosomal subunits

What is critical to the initiation of translation?

Shine=-Delgarno sequence (ribosome recognition site)

What is the ribosome recognition site?

Shine-Dealgarno sequence

Where is the shine-delgarno sequence located?

Within the 5’ end of mRNA

BLANK binds to the BLANK rRNA within the small ribosomal subunit. This allows for the start codon to be positioned correctly for binding the first tRNA.

Shine-delgarno 16s rRNA

BLANK - s Subunit has initiation factor bound, this is normally referred to as BLANK

30s - IF3

The 30s subunit has initiation factor (IF-3) bound. This does what?

Prevents agglomeration with 50s subunit

MRNA transcript has a (3’ or 5’) (Untranslated or translated) region with ribosome binding site. This is referred to as the BLANK

5’ Untranslated - Shine Delgado sequence

The Shine-Delgado sequence basic pairs with the BLANK end on the BLANK rRNA in the 50s subunit

3’ end on the 16s rRNA

Once the Shine-Delgado sequence binds, the BLANK is now bound

Small subunit

Once the small subunit is bound, BLANK is now available for recognition by tRNA carrying BLANK

- AUG start codon - formal-methionine

The first tRNA binds with the help of BLANK to the future BLANK site

IF-2 - P-site

BLANK displaces IF-3 to allow association of the BLANK subunit

IF-1 - 50s subunit

Small subunit -> BLANK -> BLANK -> Large subunit

- mRNA - tRNA

BLANK uses energy to ensure tRNA binding then disassociates as well

IF-2

BLANK = displaces IF-3 to allow association of the 50s subunit BLANK = helps tRNA bind to the future P-site then disassociates BLANK = the normal initiation factor bound to the 30s subunit

IF-1 IF-2 IF-3

What is the order of sites on the ribosome?

A -> P -> E

Where does the first tRNA start? What is the first tRNA?

The P-site - pseudomethionine

Once the first tRNA binds, where do subsequent tRNA molecules bind first?

At the A-site

BLANK help the proper functioning of elongation

Elongation factors

What are some elongation factors?

EF-Tu EF-Ts Peptides Transferase EF-G

BLANK Brings tRNA to the A site, hydrolyzes BLANK to lock correct tRNA in place.

EF-Tu GTP

BLANK recycles GTP onto BLANK and takes away spent GDP. This ensures steady supply of EEF-Tu ready to bind new tRNA

EF-Ts EF-Tu

BLANK catalyzes the addition of AA from the P-site to the A-site. This is also referred to as a BLANK. The 23s rRNA component of the BLANK subunit.

Peptidyl Transferase - Ribozyme - large subunit

Once the P-site tRNA is empty, the BLANK translocates (moves downstream by 1 codon = 3 nucleotides). This is facilitated by BLANK and uses energy

Ribosome - EF-G

Once the empty tRNA moves, full tRNA moves from BLANK to BLANK site.

Moves from A site to P site

What is the rate of Amino acids per second in a bacterial cell ribosome

16 AA / Second

How is termination brought about in the A-site?

Stop codon

Stop codons are bound by what?

Release factors (RF 1-3)

BLANK cleaves peptide bond with no new AA to attach, releasing the finished polypeptide

Peptidyl Transferase

Once the amino acid is cleaved and let loose, what two things dissociate and reset to starting conditions?

Ribosome and mRNA

Each gene in a BLANK mRNA transcript is translated independently as each gene has its own BLANK sequence and BLANK codon.

Polycistronic - Shine-dalgarno sequence - Stop codon

T/F: Multiple ribosomes can work on the same mRNA molecule simultaneously

True

Are Polysomes unique to prokaryotes>

Why can transcription and translation occur simultaneously in prokaryotes?

They lack a nucleus to separate the two processes

COMPARING TRANSLATION Simultaneous transcription and translation?

Bacteria: Yes Euk: No Archaea: Yes

COMPARING TRANSLATION Recognition of mRNA by small ribosomal subunit

Bacteria: Shine-Delgarno Eukarya: 5’ cap Archaea: Shine Delgarno

COMPARING TRANSLATION Coding of start codon

Bacteria: Formyl-Met Euk: Met Archaea: Met

COMPARING TRANSLATION Amount of translation factors

Bacteria: Fewer, less complex Eukarya: More, more complex Archaea: More, Less complex

COMPARING TRANSLATION Amount of termination factors

Bacteria = 3 Eukarya = 1 Archaea = 1

BLANK proteins help most proteins fold, there are systems. What are they?

Chaperone proteins DNA K and DNA J

DNA K and DNA J are both BLANK dependent enzymes that make sure proteins don’t fold too fast to ensure the right connections are made. If it was too fast, mistakes would be made.

ATP dependent enzymes

BLANK and BLANK fold whatever DNA K and J don’t, or if additional help is needed

GroEL and GroES

What is a great trigger for our immune system, and why?

Formyl-Met since we do not have these. This is often observed since the first codon is typically cleaved away (removed), however, bacteria tend to miss or not cleave all of their proteins.

Our white blood cells often have BLANK receptors

Formyl-Met receptors

Where do most of our proteins go through to have extra processing and to fulfill sugaring process? (Two places)

ER and Golgi

Bacteria do not have organelles, however, they under go BLANK to sugar proteins

Glycosylation

Antibiotics that inhibit protein synthesis often target what? Why is this not a great target?

70s ribosome. We have a 70s ribosome in our mitochondria, but it is housed within an organelle so small douses will not influence mitochondrial ribosomes. Large doses can hurt us.

What are some groups of antibiotics that inhibit protein synthesis?

Aminoglycosides Tetracyclines Chloramphenicol Macrolides

Tend to be bigger, not the easiest thing to get across the membrane. PICK ONE: A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides

Examples include Streptomycin, Gentamycin, Tobramycin, Neomycin PICK ONE: A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides

Obtained from Streptomyces PICK ONE: A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides and Tetracyclines

Obtained from Streptomyces Venezuelae PICK ONE: A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Chloramphenicol

Composition: Cyclohexane ring, one or more amino sugars. Binds to: 30s ribosomal subunit causing misreading of the mRNA message PICK ONE: A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides

Can be used against Gram (-) and Gram (+), used mainly against gram (-) enterics PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides

BLANK inhibit reproduction (type of medication)

Bacteriostatic

Can be toxic, causing deafness, sickness, etc. PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Aminoglycosides

Are Aminoglycosides bacteriocidal or bacterostatic ?

Bacteriocidal

Binds to the 30s ribosomal subunit, inhibits entrance of tRNAs into the A site PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Tetracyclines

Negatives: Can disrupt calcium levels, issues with veterinarian over usage, gets into the environment and can effect us PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Tetracyclines

Binds to the 50S ribosomal subunit, inhibits formation of peptide bond between amino acids (transpeptidase enzyme) PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Chloramphenicol

Negatives: used as a last resort due to severe side effect, causes aplastic anemia, depresses bone marrow function (since RBC depleted) PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Chloramphenicol

Less side effects than others, safer PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Macrolides

Examples: Erythromycin, Clarithromycin, Azithromycin

Macrolides

Binds to 50S ribosomal subunit, prevents translocation of ribosome (elongation factor: EFG) PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Macrolides

Contains lactose rings to one or more sugars PICK ONE A) Aminoglycosides B) Tetracyclines C) Chloramphenicol D) Macrolides

Macrolides

What is an example of post-translational control

Regulating enzyme activity

Series of enzymes that go to make an end product that the cell needs, end product concentration is usually the signal: (TYPE OF INHIBITION)

Feedback inhibition

Where and what binds in feedback inhibition?

The end product binds to the second site of the first enzyme (allosteric), disrupting the active site.

Feedback inhibition involves BLANK enzymes

Allosteric enzymes

BLANK: catalyzes the same reaction but has a different regulatory control

Isoenzyme

BLANK Feedback inhibition involves the use of isoenzymes which are different proteins that catalyze the same reaction but are under different regulatory controls

Concerted feedback inhibition

Concerted feedback inhibition: How do we inhibit this pathway fully?

Must inhibit all 3 of the products, only one or two will incrementally knock it down.

What are some covalent modification of enzymes ?

Addition or removal of a particular group/molecule

What are some main groups of added enzymes?

Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) Inorganic phosphate (PO4 2-) Methyl Groups (CH3)

Give an example of a covalent modification enzyme

Glutamine synthetase (GS)

Regulation at the translation level includes regulating what?

Enzyme synthesis

What are two examples of regulation at the translational level?

1. Riboswitches 2. Small RNAs (sRNA’s)

In riboswitches, BLANK is set up as a riboswitch

MRNA

BLANK region at the 5’ end of a riboswitch

Aptamer region

What is a specific secondary structure that allows for metabolite binding in riboswitches?

Aptamer region

Metabolite bound = BLANK blocked, inhibits BLANK from being recognized

- Translation blocked - inhibits Shine Delgarno

Metabolite not bound - BLANK is seen by ribosomal RNA to make what we need

Shine Delgarno Sequence

In riboswitches, mRNA contains a binding site for a specific metabolite, which when bound alters the mRNA hiding in the BLANK

Shine-Delgarno sequence

How many nucleotides are small RNA’s ?

40 - 400 nucleotides

BLANK is a regulation at a translational level, uses a complimentary sequence / pairing to its base target. Includes four different mechanisms.

Small RNA’s

What are the 4 different mechanisms for sRNAs ?

1. Hides Shine Delgarno (decrease initiation) 2. Opens up Shine Delgarno / more accessible (increase initiation) 3. Increase stability 4. Decrease stability

Regulation of transcription =

Regulating enzyme synthesis

What is a mechanism of regulating transcription?

Attenuation

T/F: Attentuation is only in prokaryotes

True

What is needed for attenuation to occur?

Transcription and translation at the same time, which can only occur in prokaryotes (no nucleus)

BLANK is a sequence, an example is the Tandem tryptophan residues located at the N-term

Leader sequence

In a leader sequence, tandem residues form at the N-term, the rest is able to form BLANK structures.

Secondary structures

Leader sequence: If tryptophan is plentiful = do not need to transcribe anymore = inhibit transcription If tryptophan is in short supply = need to transcribe more

Look above lol

Too much tryptophan = turn off transcription by having BLANK residues of tryptophan. BLANK forms further away, closer to BLANK, creating a stem loop that causes RNA polymerase to BLANK. Secondary structure int=reacts and terminates transcription

Tandem Secondary structure RNA polymerase Stop

Not enough tryptophan = BLANK has a hard time finding tRNA with tryptophan, lacking a leader sequence. Secondary structure forms BLANK since ribosome is not zipping through it. Secondary structure forms further from RNA polymerase, does not disrupt it.

Ribosome Earlier

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Exam 4 - TY Flashcards

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