Ch. 11 Gene Expression: Translation and Genetic Code Flashcards
Sickle cell anemia
First inherited human disease to be studies on a molecular level.
Discovery in 1957: normal red blood cells: Hemoglobin contains 2 alpha and beta globin chains (and the iron containing heme group). The 6th amino acid in the beta chain in a healthy hemoglobin is glutamic acid. In sickle cell disease, this is replaced by valine.
Difference between healthy and sickle shaped blood cell is based on?
One single amino acid
Proteins Structure
Complex macromolecules composed of?
20 different amino acids
Proteins are made of
Polypeptides
Polypeptide
a long chain of amino acids
Amino acids
Have a free amino group. a free carboxyl group, and a side group (R).
There are 20 different side groups, hence there are 20 different amino acids.
Different kinds of side groups
Hydrophobic or nonpolar.
Hydrophilic or polar.
Acidic.
Basic.
Amino acids are joined by?
Peptide bonds. (to make a polyPEPTIDE)
The carboxyl group of one amino acid is covalently attached to the amino group of the next amino acid.
Levels of protein structure
Primary structure - amino acid chain=polypeptide chain.
Secondary structure - 2 types, helix or beta sheet.
Tertiary structure - further folding.
Quaternary structure - several polypeptide interacting with each other. Non protein proteins can be added - heme group.
Molecular interactions determining tertiary structure (side chains)
The side chains (R) of the amino acids in the polypeptide chain react with each other. This determines (and stabilizes) the tertiary structure of a protein.
Molecular interactions determining tertiary structure (side chains)
What would happen if you exchange the amino acids? (instead of arginie you insert leucine)
Diff side chains - different interaction or possible no interaction at all
In proteins, the function is determined through?
The structure
The structure of a protein is determined through through?
The sequence of amino acids in the primary structure.
If you change this sequence, you change the structure, you change the function of the protein (or the protein loses it’s function)
Genes encode polypeptides
Classic experiments revealed that genes specify the structure of polypeptides by means of a code composed of fundamental units called codons, and that each codon is 3 nucleotides long.
The Beadle and Tatum experiment
One gene - one enzyme hypothesis.
(later restated as the one gene - one protein/polypeptide hypothesis)
Every gene carries info for 1 polypeptide
Crick and colleagues: each amino acid is specified by 3 nucleotides
(cracked the genetic code)
In the 1950s it became apparent that genes were made of DNA and the info was transcribed into mRNA, which when directed the process of polypeptide synthesis.
There are 20 diff amino acids and 4 diff nucleotides.
How does the encoding work?
Crick and colleagues: Cracking the genetic code
64
Groups of nucleotides must act as a coding unit.
How many nucleotides are in the coding unit (codon)?
20 amino acids means there must be at least 20 diff codons.
There must be at least 3 nucleotides in 1 codon (4x4x4=64)
4x4=16 but have to have 20 aa so not enough.
4x4x4=64 enough to cover the 20 aa.
4 bc each position can have A,T,C,G = 4 options.
Crick and colleagues: Cracking the genetic code
Reading frame
The coding sequence is a string of codons, read from 5’ to 3’ and it is nonoverlapping.
The beginning of the sequence is called the reading frame.
In a triplet codon there can be 3 reading frames - different results of aa from where you start.
Crick and colleagues: Cracking the genetic code
Experiment
Induce mutations that disrupt the reading frame by inserting or deleting a single base pair in a gene.
Bacteriophage T4: rII gene; mutants in that gene can only grow in E.coli B but not in E.coli k12.
Treat T4 with a chemical which causes a single bp insertion or deletion.
The reading frame will be thrown off.
Reading frame
The reading frame gets shifted with insertion and deletions. Every insertion (+) is suppressing every deletion (-). ( 1 time shift to right then back to the left, vise versa. Combination of 3 insertion and 3 deletions resorted the reading frame resulting in the wild type phenotype. (proved code is indeed 3 nucleotides)
The Genetic code is a triplet code
The triplet code is a set of 3 nucleotides (codons) that have the instructions to make a specific amino acid (or to start or stop the making of the polypeptide chain).
The Genetic code is a triplet code
64 codons
All 64 codons were deciphered by the mid-1960s.
Of the 64 triplets:
61 code for amino acids
3 triplets are “stop” signals to end translation process.
Start and stop codons
AUG - Met (initiator)
Stop codons - UAA, UAG, UGA
Properties of the genetic code
The genetic code is composed of nucleotide triplets.
The genetic code contains start and stop codons.
The genetic code is nonoverlapping.
The genetic code is degenerate (redundant).
The genetic code is no ambiguous.
The genetic code is nearly universal.
The genetic code is degenerate (redundant).
More than one codon may specify a particular amino acid (AAA and AAG both code for Lysine) (Wobble: 1st and 2nd bases of the codon follow strict rules, but the 3rd base pairs weakly with flexibility)
The genetic code is not ambigous
no codon specifies more than one amino acid; AAA codes only for Lysine, nothing else
GMOs are possible because the genetic code is universal
All organisms (from viruses to human) use the same genetic code (few minor exceptions). Insert a fluorescence gene from a jellyfish into a normal fish. Transcription and translation can happen. Jellyfish gene is expressed in the GloFish. (same genetic code so other animals can transcribe and translate genes from diff animals.
Translation
The process of transferring the info in mRNA molecules into the sequence of amino acids in polypeptide gene products.
Translation occurs
on the ribosomes
Tanslation stages
polypeptide chain initiation, elongation, and termination.
Components of tranlation
3 types of RNA (transcribed from chromosomal genes):
mRNA - carries info
tRNA (adaptor. 40-60 diff kinds) - transfer
rRNA - structural components of ribosomes (3-5)
Ribosomes
Amino-acid Activating enzymes (20): aminoacyl tRNA synthesis
More enzymes
Transfer RNAs (tRNAs)
Adapters between amino acids and the codons in mRNA molecules.
The amino acids are covalently attached to the 3’ end of the tRNA.
The anticodon of the tRNA is complementary to the codon of mRNA.
aminoacyl-tRNA synthetase
The enzyme amnioacyl-tRNA synthetase matches the right amino acid with the right tRNA (with the corresponding anti-codon)
There is at least one aminoacyl-tRNA synthetase for each of the 20 amino acids.
According the genetic code there are 64 diff tRNAs
High specificity of tRNAs
tRNA molecules must have the correct anticodon sequence.
tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase.
tRNA molecules must bind to the appropriate sites on the ribosomes during translation. (adapter b/t aa and codon)
Ribosomes
Workbench: machines and tools to make a polypeptide.
E.coli about 200,000 ribosomes, 25% dry weight of the cell.
In prokaryotes: ribosomes are distributed throughout the cell.
In eukaryotes: in the cytoplasm and/or attached to endomembrane system (rough ER)
Ribosomes makeup
50% protein, 50% RNA
2 subunits
S= Svedberg unit: measurement of sedimentation during centrifugation.
2 subunits assemble themselves during translation. Always large and small subunit.
Prokaryote and eukaryote ribosomes are very diff, important when designing drug to kill bacteria but not host.
Ribosome - prokaryotes
50S subunit. 30S subunit.
20 nm
70S ribosome
16s rRNA - can be used for identification. Not going to get extra infor from human DNA when analyzing.
Ribosome - Eukaryotes
60S subunit. 40S subunit.
24 nm
80S ribosome.
18S rRNA - don’t get extra info from bacteria DNA.
Ribosomal RNA (rRNA)
Transcribed from DNA templates.
In eukaryotes, rRNA synthesis happens in the nucleolus (a specialized region of the nucleus devoted exclusively to the synthesis of of rRNA).
RNA polymerase 1 - catalyzing synthesis.
Transcription results in rRNA precursor (primary transcript).
Undergoes further modification.
tRNA binding sites on the ribosome
E or exit tRNA binding site
P or peptidyl binding site
A or aminoacyl binding site
Translation initiation in E.coli
Initiation: means all events before the formation of a peptide bond between the 1st 2 amino acids.
Involves the 30S subunit of the ribosome, initiator tRNA (tRNA met) (start codon), mRNA, initiation factors (IF-1, IF-2, IF-3), and one molecule of GTP (energy).
1st a free 30S subunit interacts with the mRNA and the initiation factors (assembles itself onto mRNA).
Then, the 50S subunit of the ribosome joins.
Translation initiation in E.coli
Step 1
Formation of IF-2/tRNA met and IF-3/mRNA/30S subunit complexes
Translation initiation in E.coli
Step 2
The complexes formed in step 1 combine with each other, IF-1, and GTP to form the 30S initiation complex.
Translation initiation in E.coli
Step 3
After IF-3 is released, the 50S subunit joins the initiation complex; GTP is cleaved and IF-1 and IF-2 are released.
The Shine-Dalgarno Sequence
Complementary to a region of 16S rRNA (the RNA component of the 30S subunit).
mRNA and 16S rRNA will pair in that region.
Necessary for the formation of the initiation complex.
Translation Initiation in Eukaryotes
The amino group of the methionine on the initiator tRNA is not formylated.
The initiation complex forms at the 5’ terminus of the mRNA (there is no Shine-Dalgarno sequence)
The initiation complex scans the mRNA for an AUG initiation codon.
More initiation factors are binding at the 7-methyl guanosine cap at the 5’ terminus.
Polypeptide Chain Elongation
An animoacyl-tRNA binds to the A site of the ribosome.
The growing polypeptide chain is transferred from the tRNA in the P site to the tRNA in the A site by the formation of a new peptide bond.
The ribosome translocates along the mRNA to position the next codon in the A site. At the same time
-The nascent polypeptide-tRNA is translocated from the A site to the P site.
-The uncharged tRNA is translocated from the P sire to the E site. (exiting the ribosome)
(Ribosome is moving along the mRNA)
Polypeptide Chain Elongation
Steps
- codon recognition (b/t anticodon and codon) A site
- Peptide bond formation (peptide from A is put onto chain on P)
- Peptide chain is transfered to A site
- Translocation (tRNA from A site moves to P site; tRNA at P site is ejected from E site)
Ribosome ready for next aminoacyl-tRNA
Polypeptide Chain Termination
Polypeptide chain termination occurs when a chain-termination codon (stop codon) enters the A site of the ribosome
The stop codons are UAA, UAG, UGA.
When a stop codon is encountered, a release factor binds to the A site.
A water molecule is added to the carboxyl terminus of the nascent polypeptide, causing termination.
Posttranslational modifications
(polypeptide chain not a finished protein yet so has to go through modifications)
Examples:
The N-terminal amino acid is often removed or modified: in prokaryotes: formyl group or the entire formylmethionine is removed; in eukaryotes: amino group of the initial methionine residue is often removed.
Acetylation, phosphorylation
Attach carbohydrate side chains (glycoproteins) (carbohydrate sugars and proteins)
Trimming
Tagging to signal transport (postal codes)
Polypeptide chains often complexed with metals (hemoglobin)
Protein folding and unfolding
Proteins folding
Not spontaneous
Dependent on chaperones: Proteins that mediate folding process
Protein folding and unfolding
Disease of protein folding
Creutzfeldt-Jacob disease (mad cow disease)
Transmittable brain disorder in mammals
Presence of prions (misfolded proteins) in brain.
Translation and antibiotics
Effective antibiotics kill bacteria without harming the patient.
Because the process of translation is essential to all living organisms, but significantly diff between bacteria and eukaryotes, it is frequently the target of drug design.
Many antibiotics bind selectively to bacterial ribosomes and inhibit specific steps, while not affecting eukaryotic ribosomes.
For example, tetracyclines bind to the A site and block entry of tRNAs (polypeptide chain can’t grow), and streptomycin binds to the small subunit and inhibits initiation
The genetic code is
Degenerate, has 1 start codon and 3 stop codons, considered to be universal.
If there were 75 naturally occurring amino acids then what is the smallest codon size?
4
What is the name given to the 3 bases in mRNA that bind to the anticodon of tRNA to specify an amino acid placement in a protein?
codon
What is the initiator triplet in both prokaryotes and eukaryotes? What amino acid is recruited by this triplet?
AUG; methionine
The genetic code is fairly consistent among all organisms. The term often used to describe such consistency in the code?
Universal
The realtionship among a gene, mRNA, and protein
genes make mRNA, when then make proteins
If humans had 25 amino acids instead of 20, then how many aminoacyl tRNA synthetases would humans have?
25
Translation in bacterial and eukaryotic cells has many similarities, but there are also several key differences. What is one of those differences that is seen in eukaryotes?
Eukaryotes use the 5’ G-cap and Poly-A-tail on their mRNAs to initiate translation.
The primary structure of a protein is determined by
the sequence of amino acids
The set of activating enzymes that attach amino acids to the correct tRNA molecule.
Aminoacyl-tRNA synthetase
