BIOL 2500 - Topic 2 (part 8-14)

Amino acid components

1.) Central ⍺-carbon

2.) A 5’ amino group

3.) A 3’ carboxyl group

4.) An R group, which is what differentiates each amino acid from each other

Linkage of amino acids

They are joined together by covalent peptide bonds, specifically between the amino group of AA and the carboxyl group of another AA, thereby forming polypeptides

Non-polar AA

1.) Glycine

2.) Alanine

3.) Valine

4.) Proline

5.) Leucine

6.) Isoleucine

7.) Methionine

8.) Tryptophan

9.) Phenylalanine

Polar uncharged AA

1.) Serine

2.) Threonine

3.) Tyrosine

4.) Cysteine

5.) Asparagine

6.) Glutamine

Positively charged AA (basic)

1.) Lysine

2.) Arginine

3.) Histidine

Negatively charged AA (acidic)

1.) Aspartate

2.) Glutamate

Primary protein structure

The sequence of amino acids, synthesized by the ribosomes

What do ribosomes make?

They make polypeptides which then become proteins

Secondary protein structure

Local regions that fold to create alpha helices and beta-pleated sheets, as a result of hydrogen bonds, van der waals interactions, and the hydrophobic effect

R-groups of alpha-helices

1.) It depends on their location in the protein and the location of the protein itself

2.) Non-polar R-groups will usually fold into the helix, unless the protein is in a non-polar environment, in which case the R-groups will primarily be pointing outwards

R-groups of beta-pleated sheets

They R-groups are sticking right up or down, therefore they are usually the polar or charged R-groups

Tertiary structure

1.) The 3D shape of the protein, as a result of the interactions between the R-groups

2.) It is stabilized by disulfide bridges and noncovalent interactions

Significance of cysteine

It is the only amino acid that can make covalent bonds

Quaternary structure

Multi-peptide protein sequences, where each subunit in the complex is a polypeptide

Tertiary vs. quaternary structure

Most proteins only go up to tertiary structure

Beadle and Tatum

In 1942, they proposed the one-gene-one-polypeptide hypothesis, using the fungi Neurospora crassa to determine that genes controlled the production of proteins

Neurospora crassa

A haploid model organism for fungi

The genetic code

Refers to the 3 nucleotide sequences called codons transcribed from DNA, each of which code for a specific amino acid

DNA sequence =

Amino acid sequence

Synonymous codons

Refers to codons that code for the same amino acid

Genetic code is…

Degenerate/redundant

The genetic code being redundant

There are 64 codons, but only 20 amino acids

Why are there so much more codons than amino acids

This is because in addition to being degenerate, it is also non-ambiguous, such that an amino acid can be encoded by multiple codons, but each codon only codes for one amino acid

Is the genetic code overlapping or non-overlapping

It is non-overlapping, such that the genetic code is read continuously, with no gaps or pauses, and no codon gets skipped over

1 tRNA = 1 AA

1.) Each codon specifies for 1 tRNA, which brings in 1 codon

2.) Therefore, we know that ten codons will bring in 10 tRNA’s and ten amino acids

Codon vs. anticodon

They are complementary and antiparallel to each other, such that the codon goes from the 5’ to 3 direction and the anticodon goes from the 3’ to 5’ direction

Who discovered that the genetic code was non-overlapping

Crick and Brenner in 1961

Who cracked the genetic code

Nirenberg in 1961, who won a Nobel Prize for it in 1968

How did Nirenberg crack the genetic code

1.) He synthetically produced RNA and inserted it into E.coli to deduce which codons are responsible for which amino acids

2.) Usually, he would only use a specific specific sequence repeated over and over again

What does it mean when the genetic code is universal

It means that it is relatively the same in most organisms

Stop codons

1.) They do not code for amino acids and instead signal the termination of translation

2.) They are also known as nonsense codons and termination codons

Discovery of the first stop codon

It is discovered by Brenner in 1965

Nonsense mutations

Single point mutations that change a protein-coding codon to a stop codon, thereby resulting in a shortened protein

Dangers of nonsense mutations

In multiple protein-coding codons, only one mutation has to occur in certain positions in order for it to become a stop codon

tRNAs function

They bring the amino acids to the codons during translation

tRNA structure

1.) It is usually 75 nucleotides in length, with a cloverleaf shape that has 4 double-helical stems and 3 single-stranded loops

2.) It contains a CCA at the 3’ end, which is what is bound to the amino acid

3.) It also has the anticodon loop, which contains the anticodon complementary to the codon on the mRNA

t-RNAs modified nucleotides

It has modified nucleotides that allows for the formation of the wobble position at the 5’ end of the anticodon loop

Wobble position

It allows multiple tRNAs to recognize multiple codons, therefore only need one or a few tRNAs for one amino acid

Wobble position advantage

It reduces the amount of tRNAs an organism needs to make, otherwise it would need 61 different tRNAs

Charging tRNAs

tRNAs carrying an amino acid needs to be charged, which is done using aminoacyl-tRNA synthetases (there are 20 in total, one for each amino acid)

Charging tRNAs (process)

1.) The tRNA and the amino acid binds to the active site of the amino-acyl synthetase

2.) The carboxyl group of the AA then reacts with the alpha-phosphate of an ATP that comes in, thereby forming a 5’-aminoacyl-AMP, releasing a pyrophosphate in the process

3.) The hydroxyl group of the CCA sequence on the tRNA then attacks and bonds with the amino acid, releasing the AMP and forming a charged tRNA in the process

3 functions of ribosomes

1.) Bind mRNA and identify the start codon for translation

2.) Facilitate complementary base-pairing between mRNA codons and tRNA anticodons

3.) Catalyze peptide bond formation

Prokaryotic vs. eukaryotic ribosomes

They primarily differ based on the number of their ribosomal proteins and the sequence of the rRNA molecules, but they share structural homology

Structural homology of ribosomes throughout the 3 domains

Large ribosomal subunit and small ribosomal subunit, both of which are measured in Svedberg units

Svedberg unit

It is not directly proportional to weight and instead focuses more on size, such that the higher the S, the larger the molecule

tRNA binding sites of the ribosome

1.) A site (aminoacyl-tRNA binding site)

2.) P site (peptidyl site)

3.) E site (exit site)

A site

Site where incoming charged tRNA binds to

P site

It is where the peptide bond between the amino acids form and where the amino acid disconnects from the tRNA

E site

Site where deacylated (i.e. uncharged) tRNA are released from the ribosome

Extra centers of the ribosomes

1.) Decoding center

2.) Peptidyl-transferase center

Decoding center

Domain in the ribosome that ensures that only the tRNA with the right anticodon enters the A site

Peptidyl-transferase center

Part of the large ribosomal subunit that forms the peptide bonds

Initiator codon

The first amino acid is always methionine, with the initiation codon AUG

Initiator tRNAs

Both bacteria and eukaryotes have 2 kinds of tRNAs that code for methionine, one that holds the normal methionine amino acid and one that specifically holds the starting methionine

Methionine tRNAs in bacteria

tRNA^Met

tRNA^fMet

Methionine tRNAs in eukaryotes

tRNA^Met

tRNA_i^Met

Identifying the initiator codon

The initiator codon can be found in the 5’-UTR region of the mRNA, but how it is found differs between bacteria and eukaryotes

Identifying the initiator codon in bacteria

The ribosome binds to the RBS, which positions the small subunit to be in the correct spot to align the start codon into the P-site

Identifying the initiator codon in eukaryotes

The ribosome looks for the Kozak sequence, which is a sequence that contains the AUG start codon

Translation initiation in bacteria (formation of the 30S initiation complex)

1.) IF1 and IF3 bind to the A site and E site (respectively)

2.) The mRNA then binds to the 30S subunit and aligns the RBS so that the AUG is in the P site

3.) Thereby forming the 30S initiation complex

Translation initiation in bacteria (after the formation of the 30S initiation complex)

1.) IF2 brings in the starting tRNA to the P-site and then uses GTP to release the IF2

2.) The other IFs also dissociate, allowing for the 50S subunit to bind and form the full functional ribosome

Translation initiation in eukaryotes (binding of the IFs)

1.) eIF3, 1, 1A and 5 bind to the 40S subunit, bringing the starting tRNA along with it, into the P site

2.) An eIF4 complex binds to the 5’ cap and then stabilizes and unwinds parts of the 5’-UTR that may have paired up with itself

eIF1A

It binds to the A site, to prevent premature binding of the large subunit

eIF2 and eIF5

It brings in the charged starting tRNA into the P site

eIF3

It blocks the association of the large subunit

eIF4 complex

eIF4A, E and G come together to form the overall complex eIF4F

Translation initiation in eukaryotes (looking for the AUG)

1.) eIF4A uses ATP to promote the ribosome to scan for the Kozak sequence, by physically pulling the ribosome

2.) Once found, eIF4G binds to the PABP proteins that are bound to poly-A-tail to circularize the mRNA, making the overall process faster

PABP (acronym)

Poly-A binding proteins

Translation initiation in eukaryotes (IF release)

1.) The IFs dissociate, allowing for the large subunit to bind to the small subunit

2.) But the eIF4F complex remains, available for the next round of translation

Translation initiation in bacteria vs. eukaryotes

1.) Eukaryotes require a lot initiation factors

2.) Bacteria bind IF’s, mRNA, and then the starting tRNA, while eukaryotes bind IF’s, starting tRNA, and then end with the mRNA

Translation elongation in bacteria (formation of the ternary complex)

The ternary complex is when the charged tRNAs associate with the EF-Tu and GTP

Translation elongation in bacteria (adding the AA)

1.) The ternary complex enters the A site, based on the tRNA anticodon that was determined by the decoding center

2.) The binding causes the ribosome to change shape, in a way that causes the tRNA in the P-site to become displaced and lean towards the tRNA in the A site

3.) EF-Tu hydrolyzes GTP, leaving the ternary structure and then the peptidyl-transferase center forms the peptide bond

Translation elongation in bacteria (translocation)

1.) Once the peptide bonds has formed, the tRNA’s shift again, making it easier for them to move into the next site

2.) EF-G comes int the A-site and displaces the tRNA

3.) EF-G then hydrolyzes GTP, causing the ribosome to change shape and shift one codon towards the 3’ end of the mRNA

4.) The uncharged tRNA and EF-G leaves the ribosome, allowing for a new charged tRNA to enter the A site

Translation elongation in eukaryotes

It’s pretty much the same as in bacteria, but it uses different elongation factors

Translation termination

Elongation continues until a stop codon enters the A site, which then causes release factors to come in and initiate termination

Release factors

They mimic the shape of tRNA and binds to the A site, recognizing specific stop codons based on their tripeptides

Release factors in eukaryotes vs. bacteria

Bacteria: RF1 recognizes UAA/UAG and RF2 recognizes UAA/UGA

Eukaryotes eRF1 recognizes all stop codons, while eRF3 stimulates peptide release

Translation termination (process)

1.) The release factor corresponding to the stop codon binds to the A site

2.) Water enters the peptidyl-transferase center, which breaks the last bond from the tRNA in the P-site and releases the amino acid chain from the ribosome

Removal of release factors

RF3 promotes the release of RF1/RF2 and then GTP is hydrolyzed to release the RF3 and ribosome

Recycling the ribosome

The ribosome recycling factor (RRF) binds to the A site, along with IF3, to prepare for the next round of translation

Genetic variation can be caused by…

1.) Recombination

2.) Mutations

Recombination

Crossing over during meiosis

Mutations

Random changes that are not repaired

Are mutations bad?

No they are not, most mutations that occur just contribute to genetic variation

Occurence of mutations

They can either occur spontaneously or through being exposed to mutagens

Germ-line mutations

Mutations that are passed from one generation to the next

Germline mutations example

Mendel’s experiments with traits in peas

Somatic mutations

Mutations that can be passed onto subsequent generations in a cell lineage, due to mitosis, but not to an offspring

Consequences of somatic mutations

It can cause tumours

Wild-type vs. mutatant DNA sequences

Wild-type sequences are the genotype/phenotypes common in the population, while mutant sequences are those that are not common

Describing coding-sequence mutations

They are usually described based on the levels within the central dogma (i.e. amino acid level, RNA level, DNA level)

Mutations at the amino acid level

1.) Synonymous and nonsynonymous mutations

2.) Used to describe the change/effect of a mutation in the amino acid sequence

Mutations at the RNA level (use)

It is used to describe the change/effect of a mutation in the codon

Mutations at the RNA level (types)

1.) Missense

2.) Silent

3.) Nonsense

4.) Nonstop

Missense mutations

When a change in the codon occurs that also changes the amino acid (aka nonsynonymous mutations)

Silent mutations

It is when the codon changes, but it does not change the amino acid (aka synonymous mutations)

Nonsense mutations

It is when the codon changes into a stop codon

Nonstop mutations

It is when a mutation occurs that causes the stop codon to code for an amino acid

Mutations at the DNA level (use)

Used to describe the change in the nucleotide sequence

Mutations at the DNA level (types)

1.) Substitutions (transitions and transversions)

2.) Insertions/deletions (frameshift)