DNA REPLICATION AND PROTEIN SYNTHESIS FROM GENE TO PROTEIN

DNA Replication DNA Synthesis The DNA bases on each strand act as a template to synthesize a complementary strand Recall that Adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C) The process is semiconservative because each new double-stranded DNA contains one old strand (template) and one newly-synthesized complementary strand A G C T A G C T

DNA Replication DNA Polymerase DNA Polymerization Enzyme that catalyzes the covalent bond between the phosphate of one nucleotide and the deoxyribose (sugar) of the next nucleotide DNA Polymerization

DNA Replication 3’ end has a free deoxyribose 5’ end has a free phosphate DNA polymerase: can only build the new strand in the 5’ to 3’ direction Thus scans the template strand in 3’ to 5’ direction

DNA Replication Initiation Primase (a type of RNA polymerase) builds an RNA primer (5-10 ribonucleotides long) DNA polymerase attaches onto the 3’ end of the RNA primer DNA polymerase

DNA Replication Elongation DNA polymerase uses each strand as a template in the 3’ to 5’ direction to build a complementary strand in the 5’ to 3’ direction DNA polymerase

DNA Replication Elongation DNA polymerase uses each strand as a template in the 3’ to 5’ direction to build a complementary strand in the 5’ to 3’ direction results in a leading strand and a lagging strand

DNA Replication Leading Strand Topisomerase unwinds DNA and then Helicase breaks H-bonds DNA primase creates a single RNA primer to start the replication DNA polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing the matching strand in the 5’ to 3’ direction The RNA primer is degraded by RNase H and replaced with DNA nucleotides by DNA polymerase, and then DNA ligase connects the fragment at the start of the new strand to the end of the new strand (in circular chromosomes)

DNA Replication Lagging Strand Topisomerase unwinds DNA and then Helicase breaks H-bonds DNA primase creates RNA primers in spaced intervals DNA polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing the matching Okazaki fragments in the 5’ to 3’ direction The RNA primers are degraded by RNase H and replaced with DNA nucleotides by DNA polymerase DNA ligase connects the Okazaki fragments to one another (covalently bonds the phosphate in one nucleotide to the deoxyribose of the adjacent nucleotide)

DNA Replication Topoisomerase - unwinds DNA Helicase – enzyme that breaks H-bonds DNA Polymerase – enzyme that catalyzes connection of nucleotides to form complementary DNA strand in 5’ to 3’ direction (reads template in 3’ to 5’ direction) Leading Strand – transcribed continuously in 5’ to 3’ direction Lagging Strand – transcribed in segments in 5’ to 3’ direction (Okazaki fragments) DNA Primase – enzyme that catalyzes formation of RNA starting segment (RNA primer) DNA Ligase – enzyme that catalyzes connection of two Okazaki fragments

Protein Synthesis DNA provides the instructions for how to build proteins Each gene dictates how to build a single protein in prokaryotes The sequence of nucleotides (AGCT) in DNA dictate the order of amino acids that make up a protein Nucleotide sequence of His gene

Protein Synthesis DNA provides the instructions for how to build proteins Each gene dictates how to build a single protein in prokaryotes The sequence of nucleotides (AGCT) in DNA dictate the order of amino acids that make up a protein Nucleotide sequence of His gene Amino acid sequence of His protein

Protein Synthesis Protein synthesis occurs in two primary steps 1 2 mRNA (messenger RNA) copy of a gene is synthesized Cytoplasm of prokaryotes Nucleus of eukaryotes 1 mRNA is used by ribosome to build protein (Ribosomes attach to the mRNA and use its sequence of nucleotides to determine the order of amino acids in the protein) Cytoplasm of prokaryotes and eukaryotes Some proteins feed directly into rough ER in eukaryotes 2

Protein Synthesis Transcription Initiation (eukaryotes) RNA polymerase binds to a region on DNA known as the promoter, which signals the start of a gene Promoters are specific to genes RNA polymerase does not need a primer Transcription factors assemble at the promoter forming a transcription initiation complex – activator proteins help stabilize the complex Gene expression can be regulated (turned on/off or up/down) by controlling the amount of each transcription factor (eukaryotes)

Protein Synthesis Transcription Elongation 1) INITIATION Transcription Elongation RNA polymerase unwinds the DNA and breaks the H-bonds between the bases of the two strands, separating them from one another Base pairing occurs between incoming RNA nucleotides and the DNA nucleotides of the gene (template) recall RNA uses uracil instead of thymine AGTCAT UCA GUA

Protein Synthesis Transcription Elongation 5’ 3’ 5’ 3’ RNA polymerase unwinds the DNA and breaks the H-bonds between the bases of the two strands, separating them from one another. Base pairing occurs between incoming RNA nucleotides and the DNA nucleotides of the gene (template) recall RNA uses uracil instead of thymine RNA polymerase catalyzes bond to form between ribose of 3’ nucleotide of mRNA and phosphate of incoming RNA nucleotide 5’ 3’ + ATP 5’ 3’ + ADP

Protein Synthesis Transcription Elongation The gene occurs on only one of the DNA strands; each strand possesses a separate set of genes

Protein Synthesis Transcription Termination 1) INITIATION Transcription Termination A region on DNA known as the terminator signals the stop of a gene RNA polymerase disengages the mRNA and the DNA

Protein Synthesis Alternative Splicing (eukaryotes only) Exons are “coding” regions Introns are removed different combinations of exons form different mRNA resulting in multiple proteins from the same gene Humans have 30,000 genes but are capable of producing 100,000 proteins Alternative Splicing (eukaryotes only)

Protein Synthesis 1 2 mRNA mRNA copy of a gene is synthesized Transcription Translation mRNA tRNA synthesis mRNA copy of a gene is synthesized Cytoplasm of prokaryotes Nucleus of eukaryotes 1 2 mRNA mRNA is used by ribosome to build protein (Ribosomes attach to the mRNA and use its sequence of nucleotides to determine the order of amino acids in the protein) Cytoplasm of prokaryotes and eukaryotes Some proteins feed directly into rough ER in eukaryotes

Protein Synthesis Translation Transcription Translation mRNA tRNA synthesis Translation Every three mRNA nucleotides (codon) specify an amino acid

Protein Synthesis Translation tRNA have an anticodon region that specifically binds to its codon

Protein Synthesis Translation Each tRNA carries a specific amino acid Transcription Translation mRNA tRNA synthesis Translation Each tRNA carries a specific amino acid

Protein Synthesis Transcription Translation mRNA tRNA synthesis Aminoacyl tRNA synthetases attach amino acids to their specific tRNA

Protein Synthesis Translation Initiation 5’ 3’ AUGGACAUUGAACCG… Transcription Translation mRNA tRNA synthesis Translation Initiation Start codon signals where the gene begins (at 5’ end of mRNA) 5’ 3’ AUGGACAUUGAACCG… start codon

Protein Synthesis Translation Initiation Small ribosomal subunit Translation Initiation Start codon signals where the gene begins (at 5’ end of mRNA) Ribosome binding site (Shine Dalgarno sequence) upstream from the start codon binds to small ribosomal subunit then this complex recruits the large ribosomal subunit Small ribosomal subunit Ribosome Large ribosomal subunit

Protein Synthesis Translation Scanning The ribosome moves in 5’ to 3’ direction “reading” the mRNA and assembling amino acids into the correct protein large ribosome subunit small ribosome subunit

Protein Synthesis Translation Scanning The ribosome moves in 5’ to 3’ direction “reading” the mRNA and assembling amino acids into the correct protein

Protein Synthesis Translation Termination Ribosome disengages from the mRNA when it encounters a stop codon

AGCUACCAUACGCACCCGAGUUCUUCAAGC Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC

AGCUACCAUACGCACCCGAGUUCUUCAAGC Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine

AGCUACCAUACGCACCCGAGUUCUUCAAGC Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser

AGCUACCAUACGCACCCGAGUUCUUCAAGC Practice Question Translate the following mRNA sequence AGCUACCAUACGCACCCGAGUUCUUCAAGC Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser

Protein Synthesis Translation Multiple RNA polymerases can engage a gene at one time Multiple ribosomes can engage a single mRNA at one time Transcription DNA mRNAs

Protein Synthesis Eukaryotes: transcription occurs in the nucleus and translation occurs in the cytoplasm Prokaryotes: Transcription and translation occur simultaneously in the cytoplasm

RNA There are four main types of RNA: mRNA - RNA copy of a gene used as a template for protein synthesis rRNA - part of structure of ribosomes tRNA - amino acid carrier that matches to mRNA codon snRNA - found in nucleus where they have several important jobs

Web Resources Insulin Example of Protein Synthesis http://www.biotopics.co.uk/as/insulinproteinstructure.html Hemoglobin Example of Protein Synthesis http://www.biotopics.co.uk/as/insulinproteinstructure.html Collagen Example of Protein Synthesis http://www.biotopics.co.uk/JmolApplet/collagen.html

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