Protein synthesis

Big Picture Make a copy of DNA (in nucleus) Send copy out of nucleus into cytoplasm Read copy on ribosome Make a protein “Central Dogma”

Organelles and cell machinery involved in protein synthesis Nucleus & Nucleolus Ribosomes Endoplasmic Reticulum Golgi Apparatus Vesicles

The Central Dogma (aka “the big picture”) Flow of genetic information in a cell How do we move information from DNA to proteins? Transcription Translation DNA RNA PROTEIN TRAIT To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages: transcription and translation Replication

Here we go…. Stage 1: Transcription Goal Make a copy of DNA in the form of RNA Location NUCLEUS

Transcription Anatomy: DNA to mRNA Transcription Unit (GENE): segment of DNA to be transcribed transcribed DNA strand = Template Strand Only read1 strand (template strand)…make complimentary mRNA strand transcription bubble enzyme RNA Polymerase coding strand 5 3 A G C A T C G T A G A A DNA G T C A T T C T C 3 A T A C G T C G 3 T A A T 5 G G C A U C G U T C unwinding G T A G C A rewinding mRNA 5 RNA polymerase template strand

Basic structure of a protein encoding gene: DNA Promoter: identified by RNA poly…attach Gene: actual DNA turned into mRNA Terminator: RNA poly detaches

Transcription: Initiation Eukaryotes Transcription Factors Protein helpers Help RNA poly attach to promoter Looks for TATA BOX TATA Box Upstream end of promoter region

Transcription: Elongation RNA Polymerase moves along DNA, opening 10-20 bases at a time Adds new RNA Nt to the growing 3’ end RNA strand peels away & DNA helix reforms reads DNA 35

Transcription: Elongation Animations: http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html http://vcell.ndsu.edu/animations/transcription/movie-flash.htm

Question What would be the complementary RNA strand for the following DNA sequence? DNA 5’-GCGTATG-3’

Transcription: termination Eukaryotes RNA Polymerase transcribes a “signal” (AAUAAA) in terminator region of gene END

Almost done… Stage 2: Translation Goal Read mRNA and turn into a PROTEIN mRNA Goal RIBOSOME (cyto or RER) Ribosome

Ribosomes Structure ribosomal RNA (rRNA) & proteins 2 subunits large small

Ribosomes A site (accepting site) P site (protein syn site) holds tRNA carrying next amino acid to be added to chain P site (protein syn site) holds tRNA carrying growing polypeptide chain E site (exit site) empty tRNA leaves ribosome from exit site

Codon Chart mRNA is read in sets of 3 bases…CODON CODON codes for Amino Acid Start codon AUG methionine Stop codons UGA, UAA, UAG

Primary structure of a protein Messenger RNA (mRNA) A U G C mRNA start codon codon 2 codon 3 codon 4 codon 5 codon 6 codon 7 codon 1 methionine glycine serine isoleucine alanine stop codon protein Primary structure of a protein aa1 aa2 aa3 aa4 aa5 aa6 peptide bonds

Yikes…here come the details!!

Building a polypeptide 1 2 3 Initiation brings together mRNA, ribosome subunits, initiator tRNA Elongation adding amino acids based on codon sequence Termination stop codon Leu Val release factor Ser Met Met Met Met Leu Leu Leu Ala Trp tRNA C A G U A C U A C G A C A C G A C A 5' U 5' U A C G A C 5' A A A U G C U G U A U G C U G A U A U G C U G A A U 5' A A U mRNA A U G C U G 3' 3' 3' 3' A C C U G G U A A E P A 3'

Translation: Initiation Small ribosomal subunit binds to mRNA Start codon AUG= methionine AA Large ribosomal subunit binds Closes down on small

Translation: Elongation tRNA brings in A.A and it sits in A site Enzyme in the large subunit of the ribosome catalyzes a peptide bond between new AA in A site and AA in P site Ribsome moves the A site tRNA to the P site Empty tRNA at P site ejected from E site Moves codon to codon 5’ to 3’ on mRNA and builds polypeptide from N-terminus to C-terminus

Translation: Termination Stop codon reaches A site Release factors bind to A site Polypeptide released

Translation Animations http://vcell.ndsu.edu/animations/translation/movie-flash.htm

DNA 3’-TCGTACGGGATACCCAAATATCGAACTCTC-5’ Question What would be the complementary mRNA strand and amino acid sequence for the following DNA sequence? DNA 3’-TCGTACGGGATACCCAAATATCGAACTCTC-5’ mRNA: 5’AGCAUG-CCC-UAU-GGG-UUU-AUA-GCU-UGAGAG 3’ tRNA anticodons: UAC-GGG-AUA-CCC-AAA-UAU-CGA-ACU Polypeptide: met-pro-tyr-gly-phe-ile-ala mRNA: 5’AGCAUG-CCC-UAU-GGG-UUU-AUA-GCU-UGAGAG 3’ tRNA anticodons: UAC-GGG-AUA-CCC-AAA-UAU-CGA-ACU Polypeptide: met-pro-tyr-gly-phe-ile-ala

Transfer RNA (tRNA) structure “Clover leaf” structure Anticodon on “clover leaf” end Complementary to mRNA codon Amino Acid attached on 3 end

Practice all of Protein Synthesis

Compare the 3 types of RNA

Prokaryote vs Eukaryote

Ribosomes: prokaryotes vs. eukaryotes

Prokaryote Transcription initiation: RNA Polymerase recognizes promoter and binds…no transcription factors needed

Transcription in Eukaryotes 3 RNA polymerase enzymes RNA polymerase 1 only transcribes rRNA genes makes ribosomes RNA polymerase 2 transcribes genes into mRNA RNA polymerase 3 only transcribes tRNA genes each has a specific promoter sequence it recognizes

Translation in Prokaryotes Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing ribosomes read mRNA as it is being transcribed

What happens to the protein when the DNA is wrong??

Types of Mutations 2 categories: Base – pair substitution Base – pair insertions or deletions

Base-Pair Substitution Replacement of one nucleotide Silent mutations: do not present a change in protein (multiple codons for one amino acid) Missense mutations: still code for an amino acid; but the wrong amino acid. Nonsense mutation: codes for a STOP CODON – translation ends prematurely

Insertions & Deletions Additions or losses of nucleotide pairs in a gene Have deleterious effects b/c alter the “reading frame” of the genetic message = frameshift mutation Occurs whenever the insertion or deletion is NOT a multiple of three

Regulating protein synthesis

Regulating at DNA level

Histone Acetylation Histone acetylation turns genes = ON attachment of acetyl groups (–COCH3) to positively charged lysines (neutralize AA) when histones are acetylated they change shape & grip DNA less tightly = unwinding DNA transcription proteins have easier access to genes

DNA packing & methylation Chromatin modifications affect the availability of genes for transcription DNA methylation turns genes = off attachment of methyl groups (–CH3) to DNA bases (cytosine) after DNA is synthesized nearly permanent suppression of genes ex. the inactivated mammalian X chromosome

Regulating at transcription level

Post-transcriptional processing Primary transcript (pre-mRNA) eukaryotic mRNA needs work after transcription mRNA processing (making mature mRNA) mRNA splicing = edit out introns (non-coding regions) protect mRNA from enzymes in cytoplasm add 5’ G cap add 3’ Poly A tail 3' poly-A tail 3' A A A A A 5' cap mRNA 50-250 A’s P P P 5' G intron = noncoding (inbetween) sequence eukaryotic RNA is about 10% of eukaryotic gene. ~10,000 bases eukaryotic DNA exon = coding (expressed) sequence pre-mRNA primary mRNA transcript ~1,000 bases mature mRNA transcript spliced mRNA

Splicing enzymes snRNPs Splicesome several snRNPs small nuclear RNA proteins Splicesome several snRNPs recognize splice site sequence cut & paste snRNPs exon intron snRNA 5' 3' spliceosome exon excised intron 5' 3' lariat mature mRNA Prokaryotes do not have introns Not all genes have introns: histones do not Dystrophin gene (99% introns) No, not smurfs! “sNurps”

mRNA Modification Animations http://vcell.ndsu.edu/animations/mrnaprocessing/movie-flash.htm

Regulating at translation level