bio 99 midterm 4

The central dogma of Molecular biology

DNA —(Transcription)—> RNA —(Translation)—> Protein

What is a genetic code? - Codon

trinucleotide sequence that codes for an amino acid (AUG)(UAA)(UAG)(UGA)

Anticodon

triplet nucleotode sequence on tRNA that base pairs with codon on mRNA

Degenerate

multiple codons can encode the same amino acid

• consequence of there being 64 possible codons but only amino acids

Codon family

- when 4 codons specify the same amino acid

start codons

AUG —> encodes Met

Stop codons

UAA, UAG, UGA

• there are no tRNA that recognize stop codons

Open reading frame

sequence that has a start codon, long stretch of codons, then a stop codon

• find AUG to start the reading frame

Decoding the genetic code by tRNAS

codons with U or C in the 3rd position always code for the same animo acid

The wobble hypothesis

fewer than 45 transfer RNA (tRNA) molecules can decode all 61 messenger RNA (mRNA) codons****** redo this after wtaching a video

• ….

U-G Wobble Rule (Part 1)

Anticodon (3’to 5’) xxG recognizes xxC and xxU codons

U-G Wobble Rule (Part 2)

Anticodon (3’5’) xxU recognizes xxA and xxG codons

Inosine

Adenosine(A) in 1st anticodon position (5’) of tRNA is converted to inosine (I)

• can only pair with C, U, A

“I” Wobble Rule

Anticodon(3’5’) xxI recognizes xxC, xxA, and xxU codons

Wobble rules

wobble base pairing only occurs between the last 3’ position of the codon and the first 5’ position of the anticodon

Types of mutations

note* mutations happen to DNA NOT RNA, but affect mRNA sequence

• Single base substitutions

• silent

• missense

• nonsense

SIngle base substitutions

change of a single base in the DNA sequence of a gene

Silent mutation

change in codon that does not change the amino acid sequence

• GAA = Glu …. —> mutated to—> GAG = Glu

Missense mutation

change in codon that results in a different amino acid encoded

• GAA(glu) —mutated—> GAC (Asp)

Nonsense mutation

change in codon that creates an early stop codon

• GAA(glu) —mutated—> TAA (UAA) or (stop)

Frameshift mutation

insertion or deletion of nucleotides that alter the reading frame of the coding sequence

deletion mutation

deletion of one or more nucleotides

transition mutation

a purine is substituted for another purine. A → G or G → A. Most common type of mutation. Can lead to all 3 types of substitutions

in-frame insertion mutation

insertion or deletion of 3 nucleotides restores the reading frame (“in frame“)

Reversion mutations

where a mutation that is deleterious to the function of the encoded protein acquires a second mutation that allows the protein to function again

• in order for a reversion mutation to be identified, protein function must be restored (full or partially)

Reversion mutation: Missense mutation

1. (Lys to Glu) in the active site causes the protein to be non-functional. A second mutation returns the amino acid to lysine

   1. Same situation as above, but a second mutation changes the Gly to Arg, which is similar to lys and the protein is mostly functional. 

Reversion mutation: Nonsense mutation

1. (Tyr to STOP) creates a truncated protein that is non-functional. A second mutation changes STOP back into Tyr. 

• Same situation as above, but the second mutation changes STOP to Ser, allowing a full length protein and resources function.

Reversion mutation: Frameshift mutation

caused by the insertion of a single nucleotide. A second mutation causes a deletion of a single nucleotide near the site of the insertion. Protein function is restored.

Minor mutation

found in other regions of protein(not active site), substituting similar amino acid, new stop codon near the back (C-terminus), & if a reversion mutation restores reading frame mutations are close together

Catastrophic mutation

found in active site of protein, substituting dis-similar amino acid, new stop codon near the front (N-terminus), if a reversion mutation restores reading frame mutations are far apart

How does molecular nature of proteins protect from mutations

deleting an entire DNA binding domain of a protein and the transactivation domain will still work, vice versa

Biochemical basis of muatation

single base substitution —> chemical carcinogens

frameshift mutation—> intercalating agents

ionizing radiation—> x rays & large deletion

Intercalating agents

these amino acids insert themselves into the double helix causing stretching of the DNA that leads to polymerase errors during DNA replication

• acridine orange is a intercalating agent

ionizing radiation

type of radiation that causes the release of electrons from molecules, ionizing them can directly or indirectly damage DNA.

• leads to double strand DNA BREAKS

• during repair= leads to large deletion mutations

• x-rays are common form of ionizing radiation used to create mutations

large deletion

deletion of 7 or more nucleotides

Cause of Mutation: singel base substitution

Silent, missense, nonsense mutations (carcinogens)

Cause of Mutation: frameshift mutations

insertion or deletion (intercalating agents)

Cause of Mutation: Deletion mutation

loss of a large chuck of sequence (ionizing radiation)

Standard Deviation (SD)

a way to measure the amount of spread of a set of data [e.g. mean +/- SD (1 SD (plus/minus) captures about 68% of the data points)]

error bars

a graphical way to visualize the spread of data in bar graphs, without showing teh individual data points (usually based on SD but not always)

T-Test

• a way to compare the means and SDs of two data sets to assess statistically if the difference between them is significant 

P-value

• a p-value <0.05 means theres < a 5% chance (or 1 in 20 chance) that the difference is due to random chance (and 95% chance that its real)

• P value >0.05 means the difference is inconclusive (it could be real but we cannot tell)

• P-value < 0.05 means the differnece is statistically significnat

tRNA structure

• has 4 -5 arms

• anticodon arm

• amino acid arm

• others arms

tRNA structure: Anticodon arm

contains the anticodon, interacts with mRNA sequence

tRNA structure: amino acid arm

attaches to amino acid

tRNA structure: other arms

• structural, interact with ribosome, tRNA synthetase

tRNA^Tyr

tRNA that recognizes a codon for Tyrosine, but does not necessarily have a Tyrosine amino acid attached to it.

Tyr-tRNA^Tyr

tRNA that recognizes a codon for tyrosine, and is “charged” with a Tyrosine amino acid

Aminoacyl

tRNA with an amino acid attached to it

tRNA processing

• tRNA is processed post-transcriptionally into its mature form. 

1. 5’ cleavage- pre-tRNA is transcribed with extra sequence at its 5' end(leader sequence). This is cleaved off by an endonuclease called RNase P to generate the mature 5' end

2. 3’ cleavage- the extra sequence at its 3' end (the trailer) is trimmed by exonucleases (RNase D cut)

3. CCA addition- (Cytosine-Cytosine-Adenine) attached to 3’ end

4. Intron splicing- introns within anticodon loop(yellow) are excised by an endonuclease complex, then two mature exons of tRNA are ligated together to form a continuous anticodon loop

tRNA processing: Base modification

specific nucleotides are modified

tRNA processing: Cleavage

the end of transcription are removed

• 5’ cleavage

• 3’ cleavage

5’ cleavage & RNaseP

pre-tRNA is transcribed with extra sequence at its 5' end(leader sequence). This is cleaved off by an endonuclease called RNase P to generate the mature 5' end

3’ cleavage & RNase D cut

the extra sequence at its 3' end (the trailer) is trimmed by exonucleases (RNase D cut)

tRNA processing: CCA addition (amino acid)

a CCA is attached to the 3’ end of the transcript. This is what the amino acid attaches to.  

tRNA processing: Intron splicing- introns removed (only in eukarotes)

introns within anticodon loop(yellow) are excised by an endonuclease complex, then two mature exons of tRNA are ligated together to form a continuous anticodon loop

tRNA Activation or aminoacylation

• Each tRNA must have its correct amino acid attached to it. 

• 2 steps: 1) Adenylylation and 2) tRNA charging 

• both steps catalyzed by aminoacyl-tRNA synthetase (AATS)

tRNA Activation: Adenylylation

 amino acid + ATP —→ aminoacyl-AMP + PPi

• AMP attaches to carboxyl group of amino acid

(Amino Acid Activation): The enzyme binds a specific amino acid(Tyr) and an ATP molecule. It transfers the AMP portion of the ATP to the amino acid, -creates—> aminoacyl-adenylate intermediate.

• This reaction releases inorganic pyrophosphate (PPi) and stores vital energy for later use

tRNA Activation: tRNA charging

aminoacyl transferred off of AMP onto → tRNA’s CCA arm

While bound to the enzyme's active site, the appropriate tRNA molecule attaches to the complex. The enzyme transfers the activated amino acid to the 3' hydroxyl end of the tRNA, releasing the AMP molecule in the process.

second genetic code

aminoacyl-tRNA synthetase interaction with the different tRNAs

Bacteria: Initiator Met tRNA

tRNA^fMet

Bacteria: Internal Met tRNA

tRNA^Met

Bacteria: fMet

Formyl group attaches to the N-terminus of fMet, preventing fMet from being able to attack to an Amino acid in front of it. fMet can only be the first Amino acid

• cannot be added internally

• tRNA^fMet is the only tRNA that is recognized by the ribosome initiation complex

Transformylase

• converts Methionine to N-formylmethionine (fMet)

Eukaryotes: Initiator tRNA

- tRNAi^Met

• the only tRNA that is recognised by the ribosome initiation complex

Eukaryotes: Internal tRNA

- tRNA^Met

• Met is not modified

aminopeptidases

in both bacteria and eukaryotes, they often remove the N-terminus Met —> so many mature proteins DONT have Met as the first amino acid

Necessary(required)

something is necessary for a function when you need it to carry out that function

• NOT necessary= when removed there is no effect

Sufficient(all you need)

something is sufficient for a function when you can get function with only that thing

• NOT sufficient- if you add and there is no effect

Bacteria: Small subunit- 30s

• rRNA- 16s

• Protein- 21 total subunits

• Function- mRNA , tRNA assembly

Bacteria: Large subunit

• RNA- 5s and 23s 

• Protein- 36 total subunits (L1 to L36)

• Function- catalyze peptide bond formation

Eukaryotes

similar to bacteria (2 subunits) but slightly more complex, more proteins

• 40s small and 60s large

Svedberg units(S)

roughly proportional to size, but not linear relationships, based on sedimentation rates during ultracentrifugation

rRNA Processing (only in eukaryotes)

• rRNA transcribed as one long transcript (30s), then later proceeded into 16s, 23s, and 5s mature RNA (note that these do not add up to 30)

Harry Noller

Ribosome catalytic activity- what is necessary and/or sufficient for peptide bond formation? rRNA, protein, or both?

Puromychin

mimics tRNA, but binds directly to the large subunits, ribosomes will attach it to a growing polypeptide chain

• New amino acids cannot be attached to puromycin, so it terminates translation and is therefore a powerful antibiotic

• In this experiment, puromycin is just the substrate for forming peptide bonds

Ribozyme

RNA with enzymatic activity

Closest protein is 18A away from the active site of large subunit.

How manhy tRNA binding sites does the ribosome have

3! A, P, & E site

Ribosome: A site

Acceptor site, where new tRNAs enter ribosome

Ribosome: P site

Polypeptide site, where growing polypeptide chain is held

Ribosome: E site

Exit site, where tRNA are expelled after amino acid removal

Ribosome: Initiation

1. Small subunits binds mRNA

2. Small subunits bind initiator tRNA

3. Large subunits binds to the rest

Final product has= small subunits, large subunit, mRNA(start codon at p site), initiator tRNA (paired with start codon)

Shine-Dalgarno sequence

consensus sequence in front of start codon

• AGGAGGU

• Recruits mRNA to small subunit

• Directs mRNA start site to correct position on ribosome

• Base pairs with 16S rRNA on small subunits

Translation Initiation in Bacteria

Bacterial proteins involved in initiation

• IF-1 - fills A site to prevent tRNAs from binding 

• IF-2 - escorts initiator tRNA

• IF-3 - prevents large subunit from binding 

GTP hydrolysis powers formation of the initiation complex

Translation Initiation in Bacteria: step 1

1a- blocking site on a small subunit

IF-1 and IF-3 bind to 30S small subunit

• IF-1- blocks A site, prevents tRNA binding

• IF-3 blocks large subunit form binding

1b- loading mRNA

mRNA attached to 30S small subunit

• Uses shine-dalgarno sequence to position start codon right at P site 

Translation Initiation in Bacteria: step 2

Step 2- recruitment of initiator tRNA

IF-2 binds to initiator tRNA

• IF-2 is bound to GTP

• Has GTP-hydrolase activity

Initiator tRNA (fMet-tRNA^fMet)

binds to start codon

• rRNA binds to unique sequence on initiator tRNA (reason why internal tRNA^Met does not bind)

• tRNA^fMet can only bind to the P site 

• No other tRNAs can bind to P site

Translation Initiation in Bacteria: step 3

Step 3- recruitment of large subunit

• 30S changes conformation to kick out IF-3, allowing 50S large subunit to bind

• Hydrolysis of GTP to GDP causes IF-1 and IF-2 to leave

Initiation complex completed

• Both large and small subunits bound

• mRNA with start codon lined up in P site

• Initiator tRNA bound to start codon in P site of ribosome

Translation initiation in Eukaryotes

the process where ribosomes assemble on mRNA with initiator tRNA to locate the start codon (AUG)

Initiation in Eukaryotes: Step 1

Initiation in Eukaryotes: Step 2

Initiation in Eukaryotes: Step 3

Initiation in Eukaryotes: Step 4

Initiation in Eukaryotes: Step 5

Bacterial Polysome

Multiple ribosomes can translate the same mRNA simultaneously

• In bacteria, this can even happen while the mRNA is still being transcribed

• Multiple RNA polymerases can also be transcribing the same DNA at once

Eukaryotic Polysome

elF4G (part of elF4F complex) can bind to poly-A binding protein (PABP)

• Connects 5’cap to poly-A tail, forming a circle

• Facilitates translation regulation

IRES/ Internal Ribosomal Entry Site

a specialized RNA sequence that allows ribosomes to directly bind and initiate protein translation in the middle of a messenger RNA (mRNA) molecule, independently of the 5' cap structure typically required

polycistronic transcripts

a single messenger RNA (mRNA) molecule that encodes multiple different proteins.

• IRES is also used by molecular biologist to express two genes off the same transcript

• IRES makes eukaryotic polycistronic transcripts