GENES AND PROTEIN SYNTHESISChapter 7

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

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

KEY TERMS RNA transcription

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

DNA RNA

Double stranded Single stranded Adenine pairs with Thymine Adenine pairs with Uracil Guanine pairs with Cytosine Guanine pairs with Cytosine

Deoxyribose sugar 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

PromoterDNA Terminator

DNAInitiation

Elongation

TerminationGrowingRNA

RNApolymerase

Completed RNA

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

1 Codon recognition

Amino acid

Anticodon

AsiteP site

Polypeptide

2 Peptide bond formation

3 Translocation

Newpeptidebond

mRNAmovement

mRNA

Stopcodon

TRANSLATION PROCESS

Three stages Initiation Elongation Termination

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

ba

Red object = ?What molecules are present in this photo?

POLYSOME

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 lacOperon trpOperon

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 lacOperon

Regulates the production of lactose metabolizing proteins

PROKARYOTIC REGULATION trpOperon

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

(microsatellites) Sequences of long repeating base pairs TAGTAGTAGTAGTAG

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