Molecular Genetics. The Central Dogma of Biology. Gene Expression. RNA nucleotides can base pair with DNA nucleotides. Gene Expression.

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1 Molecular Genetics 2: Gene Expression Molecular Genetics Replication Precisely copying all the genetic information () S-stage of cell cycle Exact replicas passed to daughter cells Gene Expression Using a specific bit of the genetic information Make a working copy of the needed bit (gene) Take the working copy to the workshop (ribosome) Use the copied instructions to build a specific protein Feb 6, 200 What is a gene? A unit of heredity (inherited information) Structure: Short sequence of nucleotides One chromosome carries hundreds of genes Function: Order of nucleotides in determines order of amino acids in protein Each gene codes for a different polypeptide The Central Dogma of Biology molecule Gene 2 Gene Gene 3 Gene Expression Copy the recipe from the master document ( gene) in the nucleus. Use the copy of the recipe () to produce the protein on ribosomes in the cytoplasm. RNA nucleotides can base pair with nucleotides U pairs with A Heyer

2 Genes are Expressed as Proteins 2 main stages ) Transcription information copied to RNA Occurs in the nucleus 2) Translation RNA information used to construct a protein Occurs in the cytoplasm I.Transcription: to RNA A specific gene is turned on Making RNA strand from gene Starts at Promoter - region before a gene Ends at Terminator - region at the end of a gene 3 2 The flow of genetic information Figure 7.7 Transcription of a gene RNA polymerase moves along strand and builds RNA strand The stages of transcription: initiation, elongation, and termination Promoter RNA polymerase Unwound Rewound RNA Start point transcript Transcription unit Template strand of Initiation. After RNA polymerase binds to the promoter, the strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 2 Elongation. The polymerase moves downstream, unwinding the and elongating the RNA transcript Æ 3. In the wake of transcription, the strands re-form a double helix. RNA transcript 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the. Completed RNA transcript Figure 7.7. The initiation of transcription Transcription initiation complex in eukaryotic cells Promoter A region of ~25 bases upstream from the gene Contains the transcription start point Often includes a TATA box Transcriptional factors Proteins that bind to the promoter Facilitate binding of the polymerase here RNA polymerase Catalyses the synthesis of RNA 5 Æ3 Starts moving downstream from the start point Figure 7.8 TA TA AAA ATA TTTT TATA box Transcription factors RNA polymerase II Promoter 2 3 Eukaryotic promoters Start point Template strand Several transcription factors Additional transcription factors Transcription factors RNA transcript Transcription initiation complex 2. Transcriptional Elongation A Non-template strand of RNA polymerase T C C Newly made RNA A T C C A A -end A U G C A A T A G G T T Elongation RNA nucleotides T U Direction of transcription ( downstream) T G G U A A C G Template strand of Figure 7.7 RNA-polymerase II.Moves 3 Æ5 down template 2.Un-zips 0 20 bases at a time 3.Ribo-nucleotides align by base pairing with template of gene at 3 -end of elongating RNA strand 4.RNA synthesized 5 Æ3 (~60 nucleotides per sec) 5.RNA strand separates from as polymerase passes 6. re-zips Heyer 2

3 3. Termination (prokaryotes) 3. Termination (eukaryotes) Protein-coding segment Polyadenylation signal AAUAAA UTR Start UTR Clip off transcript Transcribed RNA forms GC hairpin. Hairpin causes RNA poly to fall off, terminating transcription. RNA-poly transcribes past gene and past a polyadenylation signal Enzyme recognizes the poly-a signal and clips off the RNA transcript. RNA poly continues transcribing downstream for a ways before disconnecting. Types of RNA * Each type produced by transcription from. Processing of primary transcript RNA: addition of the cap and the poly-a tail TRANSCRIPTION RNA PROCESSING Pre- A modified methyl-guanine nucleotide added to the end TRANSLATION Ribosome Polypeptide 50 to 250 adenine nucleotides added to the end by poly-a synthetase using ATPs Although each RNA molecule has only a single polynucleotide chain, it is not a smooth linear structure. Within strand complementary base pairing: Regions of complementary AU or GC pairs allow the molecule to fold on itself forming helical structures called hairpin loops. G P P P Cap UTR Protein-coding segment Start Polyadenylation signal AAUAAA AAA AAA UTR Poly-A tail Cap & tail protect from rapid degradation in the cytoplasm. Eukaryotic stay active for hours, or even days, in the cytoplasm. Prokaryotes lack cap & tail; only lasts for minutes. Figure 7.9 Processing of primary transcript RNA: RNA splicing Introns: intervening sequences non-coding regions of within a gene Must be excised from pre- before translation. Exon Intron Pre- Cap 30 3 Exon Coding segment Intron Exon Poly-A tail Introns cut out and Exons spliced together The roles of snrnps and spliceosomes in pre- splicing 2 Protein snrna RNA transcript (pre-) Exon Intron Exon 2 snrnps Spliceosome Other proteins Cap Poly-A tail UTR 46 UTR UTR: Un-Translated Region outside of coding segment. Figure 7.0 The average eukaryotic pre- is only ~5% message 3 Spliceosome components Exon Exon 2 Cut-out intron Figure 7. Heyer 3

4 Correspondence between exons and protein domains Gene Exon Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation II. Translation: the sequence of s determines the sequence of amino acids in the polypeptide Domain 2 Domain 3 Domain Codons ( words ) are RNA nucleotide triplets Each represents a specific amino acid Polypeptide Figure 7.2 Translation of RNA s II. Translation The Genetic Code On the ribosome trnas translate the sequence of 3-base nucleotide words (s) into a sequence of amino acids in a polypeptide NOTE: the is not turned into protein! specific s are associated with specific amino acids 64 s, but only 20 aa s Many s are redundant Some are start/stop signals Transfer RNAs Carry Amino Acids An aminoacyl-trna synthetase joins a specific amino acid to a specific trna Amino acid P P PAdenosine ATP Pyrophosphate P P i Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. PAdenosine 2 ATP loses two P groups and joins amino acid as AMP. Structure and symbol of transfer RNA trna molecules match amino acids to the appropriate trna anti - a triplet sequence on trna that base pairs with a 3 Phosphates trna Appropriate trna bonds covalently to amino acid, displacing AMP. PAdenosine AMP 4 Activated amino acid is released by the enzyme. P i P i Aminoacyl trna (an activated amino acid ) Figure 7.5 Heyer 4

5 Ribosomes: the site of translation The Ribosome Ribosomes are bound to ER and free in cytoplasm E site (Exit site) P site (Peptidyl-tRNA binding site) E P A A site (AminoacyltRNA binding site) Large subunit binding site Small subunit Ribosome: Made of rrna and protein (b) Schematic model showing binding sites. A ribosome has an binding site and three trna binding sites, known as the A, P, and E sites. Initiation of Translation Elongation ) Next trna binds to A site 2) Adjacent amino acids linked 3) trna in P site leaves 4) Ribosome moves to next Process continues The initiation of translation binds to ribosome at start [AUG] First trna binds to (anti to ) Polypeptide elongation Translation: Termination Fig. 7.0, -2 When ribosome reaches stop, a releasing factor (protein) binds to the instead of a trna. Polypeptide is released from trna. Ribosome then released from. Heyer 5

6 Fig. 7.0, 3-4 Fig. 7.0, 5-6 Fig. 7.0, 7-8 Fig. 7.0, 9-0 Fig. 7.0, -2 Fig. 7.0, 3-4 Heyer 6

7 Fig. 7.2 Making lots of protein Many copies of can be made from one gene Many ribosomes can make protein from the same Amplification of information allows rapid production of proteins Not done yet! Protein Shape Determines Function Post-translation modification Specific 3-D shape Shape is critical to function Denaturation = loss of shape Ë loss of function Ribbon model of lysozyme protein Regulation of Gene Expression Critical to have the right protein at the right place and right time. Initiation of transcription* Transcription factors Post-transcriptional modification Alternative splicing of RNA processing Translation Post-translational modification* * Major regulative processes (Topics for future lectures) Review Prokaryotes & Eukaryotes Heyer 7

8 Mutations Single base changes: Substitution changes one aa in protein chain Deletion or Insertion leads to Frame Shifts; changes entire aa sequence downstream Base-pair substitution Wild type A U G A A G U U U G G C U A A Protein Met Lys Phe Gly Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C Missense A U G A A G U U U G G U U A A Met Lys Phe Gly A instead of G A U G A A G U U U A G U U A A Met Lys Phe Ser Nonsense U instead of A A U G U A G U U U G G C U A A Types of mutations Met Figure 7.24 A missense point mutation: The molecular basis of sickle-cell disease Wild-type hemoglobin Mutant hemoglobin C T T C A T Normal hemoglobin Glu G A A G U A Sickle-cell hemoglobin Val In the, the mutant template strand has an A where the wild-type template has a T. The mutant has a U instead of an A in one. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Base-pair insertion or deletion Protein Wild type A U G A A G U U U G G C U A A Met Lys Phe Gly Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A U G U A A G U U U G G C U A Met Frameshift causing extensive missense U Missing A U G A A G U U G G C U A A Met Lys Leu Ala Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid A A G Missing Figure 7.23 A U G U U U G G C U A A Met Phe Gly Figure 7.25 A problem of origins: which came first? Heyer 8