Exam 2 - Lectures 7+8

mRNA translation

process where mRNA is decoded to produce proteins

Major features of mRNA translation

1. occurs in all parts of the cell cycle

2. produces functional peptides and proteins

3. most of other RNA types are not translated

4. in prokaryotes, occurs in the cytosol

5. in eukaryotes, occurs in the cytosol and on ER membrane

codons

triplets of nucleotides that code for a specific amino acid - basis of genetic code

How many standard codons are there?

64 - 4³

61 code for amino acids

3 code for stop signals

Anticodons

nucleotide sequences that are complementary to their corresponding mRNA codon sequence

tRNA

adaptor molecules that contain the anticodon and link anticodon to codon to

what direction does mRNA translation happen?

5’ to 3’

codon table

translates the genetic code into amino acids

the genetic code is degenerate which means

a single amino acid may be coded for by more than one codon (except Met and Trp)

The genetic code is nearly universal which means that

it is used by prokaryotes and eukaryotes across species

codon usage bias

codon frequencies and anticodon/tRNA frequencies vary between organisms

what causes codon usage bias?

mutations

each tRNA molecules carries an ____ and recognizes ___

carries an activated amino acid and recognizes a specific mRNA codon

How are 32 tRNA sufficient for 61 codons?

Because of wobble base pairing between tRNA anticodon and mRNA codon

where does wobble base pairing in tRNA anticodon with mRNA codon occur?

third nucleobase

what do most codes start with?

AUG - methanine

reading frame

way of dividing the sequence of nucleotides into triplets, is established by the first codon read in the sequence

if the first nucleotide is skipped, what happens?

the reading frame is altered

if there are three nucleotides skipped, what happens to the reading frame?

nothing, since amino acids are produced by nucleobase triplets

frameshift mutations

change the frame - insertions or deletions of a number of nucleotides that is not divisible by 3

slippery mRNA sequences

small stretches of codons that cause translational frame shifts (usually contains a lot of U and A nucleobases)

What causes slippery mRNA sequences?

tRNAs slipping (incorrect base pairing)

degenerate code

more than one codon codes for one amino acid - allows most minor mutations to still code for the same amino acid

NOT ALL MUTATIONS INFLUENCE PROTEINS MADE

silent mutations

from degenerate code, differ in the DNA nucleotide sequence but code for the same protein

nonsense mutations

adds stop codon early

missense mutations

change in DNA nucleotide sequence that changes the protein made

ribosomes

perform mRNA translation (protein synthesis)

five steps of mRNA translation

1. tRNAs are loaded/charged with an activated amino acid

   • tRNA is loaded with an amino acid (aka it is aminoacylated)

2. Translation initiation

   • mRNA and aminoacylate tRNA bind to the ribosome

3. Translation elongation

   • cycles of aminoacyl-tRNA binding and peptide bond formation occur until a stop codon is reached

4. Translation termination

   • mRNA and protein dissociate, ribosome recycled

5. Protein folding and posttranslational modifications

   • catalyzed by a variety of chaperones and enzymes

key players in bacterial mRNA translation

1. transfer RNA (tRNA)

2. amino acids

3. aminoacyl tRNA synthetase

4. Messenger RNA (mRNA)

5. 30S ribosome

6. 50S ribosome

7. Energy (ATP, GTP)

8. Initiation factors

9. elongation/T factors

10. Release factors

stage 1: charging tRNA with an amino acid

part a - forming aminoacyl adenylate

part b - loading the activated amino acid onto the tRNA to make a charged tRNA (called an aminoacyl-tRNA)

aminoacyl tRNA synthetases (aaRS)

enzymes that help activate amino acids and them load them onto tRNA, there are 20 different aaRS to make 32 different types of aa-tRNAs

aaRS interacts both at the amino acid arm and the anticodon region of the tRNA to provide specificity

1. the aaRS catalytic domain associates with the correct amino acid

2. the aaRS anticodon recognition domain associates with the correct tRNA anticodon sequence

ribosome

• machinery that carries out mRNA translation

• catalyzes protein synthesis using peptidyl transferase enzyme activity

• composed of a large subunit and a small subunit

• in bacteria the subunits are 50S and 30S

• in eukaryotes the subunits are 60S and 40S

• ribosome is a mixture of many different proteins as well as different rRNA molecules

• Protein subunits are stabilized by rRNA and vice-versa

3 sites of a ribosome

A site - aminoacyl

P site - peptidyl

E site - exit

A site of ribosome

site for incoming aminoacyl tRNA

P site of ribosome

holds the tRNA with the peptide attached, which is to be transferred to the new amino acid residue in the course of the peptidyl transferase reaction

E site of ribosome

the third and final binding site for the tRNA in the ribosome during translation

where does initiation initiating tRNA (fMet-tRNA) enter?

P site

where does all of the tRNAs after the initiating tRNA enter?

A site

how does the ribosome know where to bind to mRNA?

specific sequences between the promoter and the gene

• Shine-Dalgarno sequence in bacteria

• 5’ cap and Kozak sequence in eukaryotes

stage 2: translation initiation

a. the small ribosome subunit binds to multiple initiation factors, which help recruit mRNA

b. 16S rRNA binds to the ribosome and the Shine-Dalgarno sequence of mRNA to stabilize the mRNA/30S ribosome structure

c. IF2-GTP recruits tRNA (fMET) and associates with 30S ribosome/mRNA; tRNA (fMet) binds to P site

d. the large ribosome subunit associates with the IF/mRNA/ribosome/tRNA complex and releases IFs to form the full ribosome/mRNA/tRNA complex

initiation factors (IF)

proteins that bind to the small ribosome subunit during translation initiation

Stage 3: translation elongation

composed of 3 steps that all happen in the ribosome

a. decoding

b. peptide bond formation

c. translocation

Step 3a - decoding

• another charged aminoacyl-tRNA bound to GTP and elongation or T factors (Tu) enters the A site of the ribosome

• binding and peptide bond formation are accompanied by GTP→ GDP hydrolysis and release of the Tu-GDP complex from the ribosome

• Tu-GTP complex is regenerated in a process requiring other T factors and GTP

• Results in a change in the conformation of the 2nd amino-acryl tRNA that pulls its aminoacyl end into the P site

step 3b - peptide bond formation

• a peptide bond is formed between the alpha nitrogen of one amino acid and the carbonyl carbon of another amino acid

• peptidyl transferase activity that catalyzes peptide bond formation resides in the 23D rRNA (a ribozyme) rather than in any protein compartments of ribosomes

• driven by favorable entropy change

• the reaction does NOT involve chemical catalysis but may be modulated by conformation changes in the active site what can be induced by protonation

step 3c - translocation

• the ribosome shifts the next codon towards the 5’ end of the mRNA

• requires EF-G (translocase) and the energy from GTP hydrolysis

• EF-G binds the A site and displaces the peptidyl-tRNA

stage 4 - translation termination

• signaled by the presence of one of the 3 stop codons in the mRNA

• once a stop codon occupies the ribosomal A site, release factors:

  • hydrolyze the terminal peptidyl-tRNA bond to release the free polypepride and the last tRNA from the P site

  • dissociate the 70S ribosome into its 30S and 50S subunits

polysomes

ensembles of 2+ consecutive ribosomes that translate mRNA into proteins

T/F - DNA transcription and mRNA translation can occur in cells as coupled processes

TRUE

Stage 5 - protein folding and posttranslational modifications

• polypeptides leave the ribosome through an exit tunnel

• the polypeptide chain is folded and processed into its biologically active form

• proteins can fold spontaneously or have chaperonins assist

post translational modification

covalent modification of amino acids

mRNA translation is the most energy-consuming process in the cell

must be tightly controlled by ATP/GTP availability in order to maintain homeostasis

How many AP is required to make a __ long peptide?

= (# of peptides x 4)

2 ATP per aa for step 1

1 GTP for initiation

(# of aa - 1) for elongation

1 GTP for termination

DNA replication vs DNA transcription vs mRNA translation

nucleic acid (DNA or RNA) extraction

method to extract and purify nucleic acids - separating nucleic acids from other cellular components

5 basic steps of nucleic acid extraction

1. collect and resuspend the sample

2. disrupt the cellular structure to create a lysate

3. bind the nucleic acids to a purification matrix

4. wash out other cellular components from the matrix

5. elute the nucleic acid from the purification matrix

genomic DNA

total genetic info of an organism

plasmid DNA

small circular DNA usually external from genomic DNA

synthetic DNA

artificial/synthesized DNA

agarose gel electrophresis

• how to visualize nucleic acids

• nucleic acids separated based on size and length

• DNA and RNA are negatively charged so they move towards the positively charged anode

• technique to separate nucleic acids

• nucleic acids separated by applying an electric field to move the charged molecules through an agarose gel matrix

higher percentage agarose gels are better are separating __ nucleic acid fragments while lower agarose gels are better at separating ___ nucleic acid fragments

high % = smaller fragments, lower % = larger fragments

intercalating agents - ethidium bromide

• way to visualize nucleic acids

• becomes fluorescent upon binding to double stranded DNA or helical RNA

• not specific

hybridization probes (complementary fragments of DNA or RNA)

• way to visualize nucleic acids

• the probes are fluorescently labeled and bound to target DNA or RNA (binding specificity)

• better than ethidium bromide because this is

  • able to be used on RNA - while ethidium bromide binds to double stranded DNA/helical RNA, most RNA is not double helix

  • has sequence specificity

blotting

technique where biomolecules are resolved in a gel matrix, transferred to a solid support, and detected with a specific probe

Northern blot

RNA

Southern Blot

uses electrophoresis to separate DNA by size and detect it with a hybridization probe complementary to the target sequence

what technique should you use if you don’t have a lot of nucleic acid sample to test?

PCR

DNA amplification

process where DNA is enzymatically copied to generate millions of identical copies of the parent DNA molecule

reasons why you might want to amplify DNA (same reasons for nucleic acid extraction)

• forensic analysis

• genome sequencing

• paternity/maternity/ancestry testing

• medical testing

• pathogen testing

PCR - polymerase chain reaction

in vitro DNA amplification method that takes advantage of mechanisms behind cellular DNA replication

ingredients for standard PCR reaction

1. template (genomic, plasmid, or synthetic DNA)

2. primers (usually DNA fragments since they are more stable)

3. Thermostable DNA polymerase

4. Buffer (has ions needed for enzyme)

5. dNTPS (the four types of DNA nucleotides)

oligoucleotides

short, single-or double stranded DNA or RNA molecules

primer

short nucleic acid sequence that provides a starting point for DNA synthesis

For PCR, the terms primer and oligo are often used ___

interchangeably

forward primer

attaches to anti-sense strand

reverse primer

attaches to sense strand

Taq/Pfu DNA polymerase

2 different thermostable enzymes commonly used in PCR

• enables running the PCR at high temperature to completely denature DNA

PCR steps

1. denature the template DNA helix by heating the mixture over 95 C

2. anneal the DNA primer to the template by cooling to 50-70 C

3. polymerize the dNTPs to the primer using thermostable DNA polymerase by heating to 72 C (5→3 elongation by synthesis of new DNA strands)

4. allow for exponential DNA amplification by repeating the denature and anneal cycles to double the number of copies in each cycle

the number of DNA molecules produced is doubled in each PCR cycle

so after n cycles, you have 2^n copies of DNA

DNA replication vs PCR

What technique should you use if you have RNA for a template?

RT-PCR (reverse transcription)

RT-PCR

technique enabling reverse transcription of RNA to DNA using modified PCR

Ingredients for standard RT-PCR

1. RNA template

2. Primers (usually DNA)

3. Thermostable RNA dependent DNA polymerase (reverse transcriptase)

4. thermostable DNA dependent DNA polymerase (Taq or Pfu)

5. dNTPs

reverse transcriptase

enzyme used to generate complementary DNA (cDNA) from an RNA template (process is called reverse transcription)

phosphonamidite method

method of chemical synthesis

nucleoside phosphoamidites

modified nucleosides used in modern nucleic acid synthesis

purpose of the pink group in a phospoamidite

variable group that protects amino groups in the nitrogenous base from hydrolysis

purpose of green (DMT) group in a phosphoamidite

protection group for 5’ end of nucleoside until addition of next nucleoside

purpose of the purple group in a phosphoramidite

the phosphoramidite moiety

prupose of the orange group in the phsophoramide

2-cyanoethyl protecting group

purpose of the yellow group in the phosphoamidite

diisopropylamino leaving group

steps of nucleic acid synthesis by phosphonamidite

1. attach 3’ end of the protected nucleotide to a support (silica)

2. remove the 5’ DMT protecting group

3. add next protected nucleoside, which is a nucleoside phosphoramidite

4. oxidize backbone linkage

5. repeat steps 2-4

6. remove protecting groups

7. cleave from silica support

chemical synthesis of nucleic acids by the phosphoramidite method proceed in what direction?

3’ → 5’ (opposite of biological synthesis of nucleic acids)

DNA sequencing

process of determining a DNA sequence

what size bp fragments are analyzed in first generation DNA sequencing

500-1000 bp fragments

Sanger sequencing

based on random incorporation of chain-terminating dideoxynucelotides during in vitro DNA replication

ingredients for Sanger DNA

1. dideoxy or chain-terminating version of all four nucleotides (ddNTPs) each labeled with a different fluorescent dye or radiolabel

2. thermostable DNA polymerase

3. Buffer

4. primers (usually DNA)

5. dNTPs

6. template to be sequenced

why are ddNTPs chain terminating?

because they inhibit elongation by DNA pol

manual method of Sanger

separate the DNA fragments using electrophoresis to determine each nucleotide in sequence

automated method of Sanger

detect in real time by purification through column and reading fluorescence with sequencer one base at a time