From Gene to Protein How Genes Work SLIDE SHOW BY KIM FOGLIA (modified) All Blue edged slides are Kim’s (hyperlinks may have been added) How Genes Work

Essential knowledge 3 .A.1: DNA, and in some cases RNA, is the primary source of5. DNA replication ensures continuity of hereditary information. c. Genetic information flows from a sequence of nucleotides in a gene to a sequence of amino acids in a protein. 1. The enzyme RNA polymerase reads the DNA molecule in the 3’ to 5’ direction and synthesizes complementary mRNA molecules that determine the order of amino acids in the polypeptide. 2. in eukaryotic cells the mRNA transcript undergoes a series of enzyme-regulated modifications. To foster student understanding of this concept, instructors can choose an illustrative example such as: Addition of a poly-A tail. Addition of a GTP cap Excision of introns 3. Translation of the mRNA occurs in the cytoplasm on the ribosome. 4. In prokaryotic organisms, transcription is coupled to translation of the message. Translation involves energy and many steps, including initiation, elongation, and termination. X The details and names of the enzymes and factors involved in each of these steps are beyond the scope of the course and the AP Exam. The salient features include: i. The mRNA interacts with the rRNA of the ribosome to initiate translation at the (start) codon. ii. The sequence of nucleotides on the mRNA is read in triplets iii. Each codon encodes a specific amino acid, which can be deduced by using a genetic code chart. Many amino acids have more than one codon. iv. tRNA brings the correct amino acid to the correct place on the mRNA. v. The amino acid is transferred to the growing peptide chain. vi. The process continues along the mRNA until a “stop” codon is reached. vii. The process terminates by release of the newly synthesized peptide/protein. d. Phenotypes are determined through protein activities

Essential knowledge 4 .A.1: The subcomponents of biological molecules and their sequence determine the properties of that molecule. a. Structure and function of polymers are derived from the way their monomers are assembled. 1. in nucleic acids, biological information is encoded in sequences of nucleotide monomers. Each nucleotide has structural components: a five carbon sugar (deoxyribose or ribose), a phosphate and a nitrogen base (adenine, thymine, guanine, cytosine, or uracil). DNA and RANA differ in function and differ slightly in structure, and these structural differences account for the differing functions [See also 1.D.1, 2.A.3, 3.A.1] b. Directionality influences structure and function of the polymer. 1. Nucleic acids have ends, defined by the 3’ and 5’carbons of the sugar in the nucleotide, that determine the direction in which complementary nucleotides are added during DNA synthesis and the direction in which transcription occurs (from 5’ to 3’). [See also 3.A.1] 2. Proteins have an amino (NH3) end and a carboxyl (COOH) end, and consist of a linear sequence of amino acids connected by the formation of peptide bonds by dehydration synthesis between the amino and carboxyl groups of adjacent monomers. LO 4.1 The student is able to explain the connection between the sequence and the subcomponents of a biological polymer and its properties [See SP 7.1] LO 4.2 The student is able to refine representation and models to explain how the subcomponents of a biological polymer and their sequence determine the properties of that polymer [See SP 1.3] SP1. The student can use representation and models to communicate scientific phenomena and solve scientific problems. SP 7. The student is able to connect and relate knowledge across various scales, concepts and representations in and across domains.

DNA gets all the glory, but proteins do all the work! The “Central Dogma” 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 DNA gets all the glory, but proteins do all the work! replication

from DNA nucleic acid language to RNA nucleic acid language Transcription from DNA nucleic acid language to RNA nucleic acid language Transcription animation

DNA RNA RNA ribose sugar N-bases single stranded lots of RNAs uracil instead of thymine U : A C : G single stranded lots of RNAs mRNA, tRNA, rRNA, siRNA… transcription DNA RNA

3 KINDS OF RNA HELP WITH INFO TRANSFER FOR PROTEIN SYNTHESIS RIBOSOMAL RNA (rRNA) Made in nucleolus 2 subunits (large & small) Combine with proteins to form ribosomes Bacterial ribosomes different size than eukaryotic ribosomes Evidence for ENDOSYMBIOTIC THEORY Medically significant-some antibiotics target bacterial ribosomes w/o harming host rRNA and t-RNA images from Image from: Biology; Miller and Levine; Pearson Education publishing as Prentice Hall; 2006 mRNA image from http://wps.prenhall.com/wps/media/tmp/labeling/1140654_dyn.gif

3 KINDS OF RNA HELP WITH INFO TRANSFER FOR PROTEIN SYNTHESIS TRANSFER RNA (tRNA) ANTICODON sequence matches CODON on mRNA to add correct amino acids during protein synthesis AMINOACYL-tRNA SYNTHETASE Enzyme attaches a specific amino acid using energy from ATP http://www-math.mit.edu/~lippert/18.417/lectures/01_Intro/

3 KINDS OF RNA HELP WITH INFO TRANSFER FOR PROTEIN SYNTHESIS MESSENGER RNA (mRNA) carries code from DNA to ribosomes

Transcription Making mRNA transcribed DNA strand = template strand untranscribed DNA strand = coding strand same sequence as RNA synthesis of complementary RNA strand transcription bubble enzyme RNA polymerase coding strand 3 A G C A T C G T 5 A G A A A G T C T T C T C A T A C G DNA T 3 C G T A A T 5 G G C A U C G U T 3 C unwinding G T A G C A rewinding mRNA RNA polymerase template strand build RNA 53 5

RNA polymerases 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

Eukaryotic transcription Which gene is read? Promoter region binding site before beginning of gene TATA box binding site binding site for RNA polymerase & transcription factors Enhancer region binding site far upstream of gene turns transcription on HIGH

Transcription Factors Eukaryotic transcription Transcription Factors Initiation complex transcription factors bind to promoter region suite of proteins which bind to DNA hormones? turn on or off transcription triggers the binding of RNA polymerase to DNA

Matching bases of DNA & RNA Match RNA bases to DNA bases on one of the DNA strands C U G A G U G U C U G C A A C U A A G C RNA polymerase U 5' A 3' G A C C T G G T A C A G C T A G T C A T C G T A C C G T

Post-transcriptional processing mRNA’s require EDITING before use primary transcript = pre-mRNA mRNA splicing INTRONS are removed (in between) EXONS stay in message (expressed) make mature mRNA transcript Image by Riedell

Starting to get hard to define a gene! Alternative splicing Alternative mRNAs produced from same gene when is an intron not an intron… different segments treated as exons Starting to get hard to define a gene!

EX: antibody production Immune system needs to be able to make a huge number of different antibodies to match new and different invaders Allows cell to use “old gene” in “new ways” Able to respond to changing environment http://www.sciencegeek.net/Biology/review/graphics/Unit8/Antibody.gif http://1.bp.blogspot.com/_bjeC1CyB28w/TOijgcetVjI/AAAAAAAAAvo/0hPZmygfSZo/s1600/colourful_cartoon_germs_bumper_sticker-p128396365988627252trl0_400.jpg

mRNA EDITING snRNPs Spliceosome PROCESSING RNA SPLICEOSOMES small nuclear RNA’s proteins Spliceosome several snRNPs recognize splice site sequence cut & paste gene THIS BREAKS THE RULES! ALL ENZYMES ARE PROTEINS???? RIBOZYMES-RNA molecules that function as enzymes (In some organisms pre-RNA can remove its own introns)

More post-transcriptional processing Need to protect mRNA on its trip from nucleus to cytoplasm enzymes in cytoplasm attack mRNA protect the ends of the molecule add 5 GTP cap add poly-A tail longer tail, mRNA lasts longer: produces more protein eukaryotic RNA is about 10% of eukaryotic gene. A 3' poly-A tail mRNA 5' 5' cap 3' G P 50-250 A’s

Figure 12–18 Translation Section 12-3

from nucleic acid language to amino acid language Translation from nucleic acid language to amino acid language 2007-2008

The code Code for ALL life! Code is redundant Start codon Stop codons strongest support for a common origin for all life Code is redundant several codons for each amino acid 3rd base “wobble” Why is the wobble good? Strong evidence for a single origin in evolutionary theory. Start codon AUG methionine Stop codons UGA, UAA, UAG

Transfer RNA structure “Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3 end

tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA Loading tRNA Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNA bond requires energy ATP  AMP bond is unstable so it can release amino acid at ribosome easily The tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome. Trp C=O Trp Trp C=O OH H2O OH O C=O O activating enzyme tRNATrp A C C U G G mRNA anticodon tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA

Protein synthesis/quiz Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon organelle or enzyme? Structure ribosomal RNA (rRNA) & proteins 2 subunits large small E P A

Ribosomes A site (aminoacyl-tRNA site) P site (peptidyl-tRNA site) translation Ribosomes A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to be added to chain P site (peptidyl-tRNA site) holds tRNA carrying growing polypeptide chain E site (exit site) empty tRNA leaves ribosome goes to get recharged Met U A C 5' A U G 3' E P A

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

start of a secretory pathway Protein targeting Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc… Signal peptide address label start of a secretory pathway

POST-TRANSLATIONAL MODIFICATIONS Some amino acids modified by addition of sugars, lipids, phosphate groups, etc Enzymes can modify ends, cleave into pieces join polypeptide strands (4’ structure) Ex: Made as proinsulin then cut Final insulin hormone made of two chains connected by disulfide bridges http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin.html

Post Translational regulation UBIQUITIN - “death tags” for proteins PROTEASOMES- recognize tags and destroy proteins http://www.caltagmedsystems.co.uk/information/wp-content/uploads/UPS.png

Can you tell the story? RNA polymerase DNA amino acids tRNA pre-mRNA exon intron tRNA pre-mRNA 5' GTP cap mature mRNA aminoacyl tRNA synthetase poly-A tail 3' large ribosomal subunit polypeptide 5' tRNA small ribosomal subunit E P A ribosome

Protein Synthesis in Prokaryotes Bacterial chromosome Protein Synthesis in Prokaryotes Transcription mRNA Psssst… no nucleus! Cell membrane Cell wall 2007-2008

Prokaryote vs. Eukaryote genes Prokaryotes DNA in cytoplasm circular chromosome naked DNA no introns Eukaryotes DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons Walter Gilbert hypothesis: Maybe exons are functional units and introns make it easier for them to recombine, so as to produce new proteins with new properties through new combinations of domains. Introns give a large area for cutting genes and joining together the pieces without damaging the coding region of the gene…. patching genes together does not have to be so precise. introns come out! intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence

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

Translation: prokaryotes vs. eukaryotes SEE PROCESSING VIDEO Translation: prokaryotes vs. eukaryotes Differences between prokaryotes & eukaryotes time & physical separation between processes takes eukaryote ~1 hour from DNA to protein no RNA processing

COMPLETING PROTEINS POLYRIBOSOMES (POLYSOMES) Numerous ribosomes translate same mRNA at same time 3-D folding (1’, 2’, 3’ structure) Chaparonins help fold into 3-D shape