Chapter 7

GENES AND PROTEIN SYNTHESIS

ONE GENE-ONE POLYPEPTIDE HYPOTHESIS

 DNA contains all of our hereditary information  Genes are located in our DNA  ~25,000 genes in our DNA (46 chromosomes)  Each Gene codes for a specific polypeptide

MAIN IDEA

 Central Dogma  Francis Crick (1956)

OVERALL PROCESS

 Transcription  DNA to RNA  Translation  Assembly of amino acids into polypeptide  Using RNA

DNA molecule DNA strand Gene 1 TRANSCRIPTION RNA TRANSLATION Codon Gene 2 Polypeptide Amino acid Gene 3

KEY TERMS

         RNA transcription  Initiation, Elongation, Termination TATA box Introns, Exons mRNA, tRNA, rRNA Translation Ribosome Codon Amino Acids Polypeptide

DNA Double stranded Adenine pairs with Thymine Guanine pairs with Cytosine Deoxyribose sugar RNA Single stranded Adenine pairs with Uracil Guanine pairs with Cytosine Ribose sugar

DNA TO PROTEIN

 Protein is made of amino acid sequences  20 amino acids  How does DNA code for amino acid?

GENETIC CODE

 Codon  Three letter code  5’ to 3’ order  Start codon  Stop codon  AA are represented by more than one codon  61 codons that specify AA

AMINO ACIDS

 Abbreviated  Three letters

TRANSCRIPTION

 DNA to RNA  Occurs in nucleus  Three process  Initiation  Elongation  Termination

RNA polymerase DNA of gene Promoter DNA Initiation Terminator DNA Elongation Termination Growing RNA Completed RNA RNA polymerase

INITIATION

 RNA polymerase binds to DNA  Binds at promoter region  TATA box  RNA polymerase unwinds DNA  Transcription unit  Part of gene that is transcribed

INITIATION

 Transcription factors bind to specific regions of promoter  Provide a substrate for RNA polymerase to bind beginning transcription  Forms transcription initiation complex

ELONGATION

       RNA molecule is built  RNA polymerase Primer not needed 5’ to 3’ 3’ to 5’ DNA is template strand Coding strand  DNA strand that is not copied Produces mRNA  Messenger RNA DNA double helix reforms

TERMINATION

 RNA polymerase recognizes a termination sequence – AAAAAAA  Nuclear proteins bind to string of UUUUUU on RNA  mRNA molecule releases from template strand

POST-TRANSCRIPTIONAL MODIFICATIONS

    Pre-mRNA undergoes modifications before it leaves the nucleus Poly(A) tail  Poly-A polymerase  Protects from RNA digesting enzymes in cytosol 5’ cap   7 G’s Initial attachment site for mRNA’s to ribosomes Removal of introns

SPLICING THE PRE-MRNA

 DNA comprised of  Exons – sequence of DNA or RNA that codes for a gene  Introns – non-coding sequence of DNA or RNA  Spliceosome  Enzyme that removes introns from mRNA

SPLICING PROCESS

 Spliceosome contains a handful of small ribonucleoproteins  snRNP’s (snurps)  snRNP’s bind to specific regions on introns

ALTERNATIVE SPLICING

 Increases number and variety of proteins encoded by a single gene  ~25,000 genes produce ~100,000 proteins

TRANSLATION

 mRNA to protein  Ribosomes read codons  tRNA assists ribosome to assemble amino acids into polypeptide chain  Takes place in cytoplasm

TRNA

 Contains  triplet anticodon  amino acid attachment site  Are there 61 tRNA’s to read 61 codons?

TRNA: WOBBLE HYPOTHESIS

   First two nucleotides of codon for a specific AA is always precise Flexibility with third nucleotide Aminoacylation – process of adding an AA to a tRNA   Forming aminoacyl-tRNA molecule Catalyzed by 20 different aminoacyl-tRNA synthetase enzymes

RIBOSOMES

 Translate mRNA chains into amino acids  Made up of two different sized parts  Ribosomal subunits (rRNA)  Ribosomes bring together mRNA with aminoacyl-tRNAs  Three sites  A site - aminoacyl  P site – peptidyl  E site - exit

TRANSLATION PROCESS  Three stages  Initiation  Elongation  Termination

mRNA movement Polypeptide P site mRNA Amino acid A site Anticodon 1 Codon recognition Stop codon New peptide bond 2 Peptide bond formation 3 Translocation

INITIATION

      Ribosomal subunits associate with mRNA Met-tRNA (methionine)  Forms complex with ribosomal subunits Complex binds to 5’cap and scans for start codon (AUG) – known as scanning Large ribosomal subunit binds to complete ribosome Met-tRNA is in P-site Reading frame is established to correctly read codons

ELONGATION

 Amino acids are added to grow a polypeptide chain  A, P, and E sites operate  4 Steps

TERMINATION

   A site arrives at a stop codon on mRNA  UAA, UAG, UGA Protein release factor binds to A site releasing polypeptide chain Ribosomal subunits, tRNA release and detach from mRNA

a

POLYSOME

b

Red object = ?

What molecules are present in this photo?

PROKARYOTIC RNA TRANSCRIPTION/TRANSLATION  Throughout cell  Single type of RNA polymerase transcribes all types of genes  No introns  mRNA ready to be translated into protein  mRNA is translated by ribosomes in the cytosol as it is being transcribed

REVIEW

 What is a gene?

 Where is it located?

 What is the main function of a gene?

 Do we need our genes “on” all the time?

 How do we turn genes “on” or “off”?

REGULATING GENE EXPRESSION

    Proteins are not required by all cells at all times Regulated Eukaryotes – 4 ways    Transcriptional (as mRNA is being synthesized) Post-transcriptional (as mRNA is being processed) Translational (as proteins are made)  Post-translational (after protein has been made) Prokaryotes  

lac

Operon

trp

Operon

TRANSCRIPTIONAL REGULATION

   Most common DNA wrapped around histones keep gene promoters inactive Activator molecule is used (2 ways)   Signals a protein remodelling complex which loosen the histones exposing promoter Signals an enzyme that adds an acetyl group to histones exposing promoter region

TRANSCRIPTIONAL REGULATION

 Methylation     Methyl groups are added to the cytosine bases in the promoter of a gene (transcription initiation complex) Inhibits transcription – silencing Genes are placed “on hold” until they are needed E.g. hemoglobin

AGOUTI MICE

POST TRANSCRIPTIONAL REGULATION

  Pre-mRNA processing  Alternative splicing Rate of mRNA degradation     Masking proteins – translation does not occur  Embryonic development Hormones - directly or indirectly affect rate Casein – milk protein in mammary gland When casein is needed, prolactin is produced extending lifespan of casein mRNA

TRANSLATIONAL REGULATION

 Occurs during protein synthesis by a ribosome  Changes in length of poly(A) tail  Enzymes add or delete adenines  Increases or decreases time required to translate mRNA into protein  Environmental cues

POST-TRANSLATIONAL REGULATION

 Processing  Removes sections of protein to make it active  Cell regulates this process (hormones)  Chemical modification  Chemical groups are added or deleted  Puts the protein “on hold”  Degradation  Proteins tagged with ubiquitin are degraded  Amino acids are recycled for protein synthesis

PROKARYOTIC REGULATION



lac

Operon  Regulates the production of lactose metabolizing proteins

PROKARYOTIC REGULATION



trp

Operon  Regulates the expression of tryptophan enzymes

CANCER

 Lack regulatory mechanisms  Mutations in genetic code (mutagens)  Probability increases over lifetime  Radiation, smoking, chemicals  Mutations are passed on to daughter cells  Can lead to a mass of undifferentiated cells (tumor)  Benign and malignant  Oncogenes  Mutated genes that once served to stimulate cell growth  Cause undifferentiated cell division

CANCER

GENETIC MUTATIONS

 Positive and negative  Natural selection – evolution  Cancer –death  Small-Scale – single base pair  Point mutations  Substitution, insertion/deletion, inversion  Large-Scale – multiple base pairs

SMALL-SCALE MUTATIONS

 Four groups  Missense, nonsense, silent, frameshift  Lactose, sickle cell anemia  SNPs – single nucleotide polymorphisms  Caused by point mutations

MISSENSE MUTATION

 Change of a single base pair or group of base pairs  Results in the code for a different amino acid  Protein will have different sequence and structure and may be non-functional or function differently

NONSENSE MUTATION

 Change in single base pair or group of base pairs  Results in premature stop codon  Protein will not be able to function

SILENT MUTATION

 Change in one or more base pairs  Does not affect functioning of a gene  Mutated DNA sequence codes for same amino acid  Protein is not altered

FRAMESHIFT MUTATION

   One or more nucleotides are inserted/deleted from a DNA sequence Reading frame of codons shifts resulting in multiple missense and/or nonsense effects Any deletion or insertion of base pairs in multiples of 3 does not cause frameshift

LARGE-SCALE MUTATIONS

 Multiple nucleotides, entire genes, whole regions of chromosomes

LARGE-SCALE MUTATIONS

 Amplification – gene duplication  Entire genes are copied to multiple regions of chromosomes

LARGE-SCALE MUTATIONS

 Large-scale deletions  Entire coding regions of DNA are removed  Muscular Dystrophy

LARGE-SCALE MUTATIONS

 Chromosomal translocation  Entire genes or groups of genes are moved from one chromosome to another  Enhance, disrupt expression of gene

LARGE-SCALE MUTATIONS

 Inversion  Portion of a DNA molecule reverses its direction in the genome  No direct result but reversal could occur in the middle of a coding sequence compromising the gene

LARGE-SCALE MUTATIONS

 Trinucleotide repeat expansion  Increases number of repeats in genetic code  CAG CAG CAG CAG CAG CAG CAG CAG  Huntingtons disease

CAUSES OF GENETIC MUTATIONS

 Spontaneous mutations  Inaccurate DNA replication  Induced mutations  Caused by environmental agent – mutagen  Directly alter DNA – entering cell nucleus  Chemicals, radiation

CHEMICAL MUTAGENS

 Modify individual nucleotides   Nucleotides resemble other base pairs  Confuses replication machinery – inaccurate copying  Nitrous acid Mimicking DNA nucleotides  Ethidium bromide – insert itself into DNA

RADIATION - LOW ENERGY

  UV B rays Non-homologous end joining  Bonds form between adjacent nucleotides along DNA strand   Form kinks in backbone Skin cancer

RADIATION – HIGH ENERGY

    Ionizing radiation – x-ray, gamma rays Strip molecules of electrons Break bonds within DNA  Delete portions of chromosomes Development of tumors

MUTATION IN PROKARYOTES

 DNA is mostly coding sequences  Mutation is harmful – superbugs

GENOMES AND GENE ORGANIZATION

 Human Body  22 autosomal chromosomes  1 pair of each sex chromosome (XX, YY)

GENOMES AND GENE ORGANIZATION

 Components  VNTR’s– variable number tandem repeats  Sequences of long repeating base pairs  TAGTAGTAGTAGTAG (microsatellites)  LINEs – long interspersed nuclear elements  SINEs – short interspersed nuclear elements  Transposons – small sequences of DNA that move about the genome and insert themselves into different chromosomes  Pseudogene – code is similar to gene but is unable to code for protein

VIRUSES

 Not alive but can replicate themselves  Contain  DNA or RNA  Capsid – protein coat  Envelope – cell membrane

VIRUS

 4000 species of virus have been classified

REPLICATION

 DNA  Transcription and translation  RNA (retrovirus)  Uses reverse transcriptase – enzyme  Uses cells parts to make a single strand of DNA and then makes a complementary strand from that copy  Integrase – incorporates into our genetic code

VIRUS AS VECTORS

 Transduction  Using a virus vector to insert DNA into a cell or bacterium