From Gene to Protein

Transcription

The Genetic Code

Translation

The Complex Proteome

Applied Lecture

Applied Lecture

Reading the Blueprint

DNA Molecule

Contains genes that code for proteins or RNA molecules, that have various cell functions

Information is stored in DNA of a cell

Stored information is interpreted and transmitted for cellular processes

Genes

Sections of DNA molecules, that contain formation that is transcribed into RNA copy

Either stays as RNa, or is further translated into mRNA, and makes up amino acid sequence, to make up proteins

Central Dogma

Process of copying and interpreting genes into proteins

Information that is stored in DNA, specifies sequence of amino acids in protein

DNA copied multiple times into mRNA

mRNA translated from nucleotide to protein

Gene Expression

Trasncription of information encoded in DNA to RNA

Transcription in Prokaryotes

DNA Transcribed from Template Strand

Nucleotide sequence contained in gene, will determine sequence of nucleotides contained in RNA transcript

RNA strand is same as template strand, due to base-pairing rule; except U replaces T

DNA Transcription Start & End Site

RNA polymerase attaches to DNA promoter regions

Promoter regions show transcription starting point

Transcribes in 3'-5' direction

Synthesized from 5'-3' direction

Terminator stops transcription

Transcription Initiation

Nucleotide sequence TATAAT is promoter sequence

Consensus sequence

Nucleotide sequence in DNA found at site

Also have TTGCCA

Enhance transcription rate

Transcription requires RNA polymerase and sigma factor proteins

Bind promoter region of DNA

Pair binds to create holoenzyme

Binds to and unwinds double-stranded DNA helix

RNA Polymerase

Elongates the forming RNA transcript

Separates DNA double helix

Ribonucleotides enter, assemble on DNA template strand

Passes template DNA through channel, transcribe template strand into complementary RNA

Restores DNA back into double helix

RNA Transcription Elongates at 3' End

Phosphate bond energy of incoming ribonucleoside triphosphate

Drive high energy reaction process

Required to create phosphodiester bond between incoming nucleotide and growing RNA transcript

Release and cleavage of pyrophosphate (phosphate-phosphate) group during phosphodiester bond formation, renders this polymerization reaction

RNA transcript elongation irreversible

Transcription Stopping

Termination sequence at 3' end

Requires nucleotide terminator

Releases RNA sequence

Releases transcription complex

Termination of Transcription

Rho-independent terminator

Inverted nucleotide repeat sequence

Fold back on themselves to form G-C hairpin loop along mRNA strand

Rho-dependent terminator

Rho factor - protein

Bind to and use ATP energy to move along RNA transcript while unwinding it from DNA template

Destabalize interaction between DNA and RNA template

Release of transcript & transcription complex

Transcription and mRNA Processing in Eukaryotes

Transcription in Eukaryotes

General Transcription Factors

Mediate binding of RNA polymerase to promoter

Initiate transcription

RNA Polymerase I

Transcribes genes for rRNA

RNA Polymerase III

Transcribes genes for tRNA

RNA Polymerase II

Transcribes mRNA

Template for production of protein molecules

RNA polymerase produces complementary, antiparallel strand of RNA

5' Cap

Post-transcriptional modifications

Add 5' cap and 3' poly (A) tail

5' end gets attachment of guanosine to mRNA through 5'-5' triphosphate linkage

7-methylguanosine

Adenine nucleotides added to 3' end is poly(A) tail

Follows polyadenylation signal sequence (AATAAA)

Termination

Poly(A)-dependent mechanisms

3' end modified by polyadenylation

Termination

similar to Rho-dependent factor

Termination

Similar to Rho-independent factors

terminated after termination signal

Processing RNA

RNA Splicing and Spliceosome

Amino acids that make up proteins, but do not code for anything

Introns

Exons

Sequence of amino acids in protein

Intervening sequence

Removed

Joined

spliced

Short nucleotide at each end of intron

Catalyzed by spliceosomes

Composed of 5 nuclear ribonucleoproteins (snRNP)

Recognizes complimentary base pairing

Finds splice site

Catalyzes reaction with hydroxyl group

Cut at 5' end forms a loop

3' end has acceptor site

Defining a Codon

Gamow

One nucleotide coded for one amino acid

1-base code not enough to code all 20 amino acids

2-base code also not enough

4x1=4

4x4=16

3-base code

4x4x4=64

More than enough, but accommodates 20 amino acids

Standard Code

Codons written in 5'-3' direction

Non-template strand is coding strand

Same as RNA except for U replaces T in RNA

AUG

Start Codon

Methionine

UGG

Tryptophan

Redundancy

Serine

4 triplets

Unique condon triplets

Never codes for more than one amino acid

Stop Codon

do not code

Reading Frames

Open Reading Frame

Continuous sequence of a gene, begins with triplet start codon, ends with triplet stop codon

Inserting Nucleotides

When adding or subtracting nucleotide, it changes codon sequence, resulting in changes to amino acids

Frameshift Mutations

Add or remove 2 nucleotides

Produce Alternate Proteins

First/Second/Third Reading Frames

Coding region of a gene

mRNA can be read from 1st, 2nd, 3rd nucleotide

Removing 3 Nucleotides

No frameshift

Protein still conserved

same for adding 2 nucleotides

RNA Splicing & Spinal Muscular Atrophy

Non-mature mRNA has exon and intron

Splicing Fails

Errors in splicing

Missing sequences

Exon skipping

Intron retention

Not producing full length protein

SMA

Onset before 6mths

Lifespan 2yrs

Affects motor movement

Skeletal muscles die

Neuron degeneration

Autosomal recessive disorder

Chromosome 5

Delta 7

SMN1 Mutations

8 exons (9 technically)

Delta 7 has 7 exons

Missing amino acid

Ribosomes don't translate nucleotide

SMA2

More = longer life expectancy

Backup for SMA1

Splicing

Need high number

Position 6, T

Splicing

Position 6, C

Cut at 7

Skipped at 7

Gene Therapy

Add SMN1

Molecular Therapy

Stop SMN2 exton 7 skipping

Molecular Components of Translation

Process of Translation

Components

Cellular components read genetic message in mRNA,

Translate into primary amino acid

Initiation

Elongation

tRNA

mRNA genetic message to polypeptide

Cytoplasmically situated amino acids -> Transfer amino acids -> Ribosome

Clover-Leaf

Hydrogen bonding between complementary nucleotide bases

4 double-helical segments and 3 loops

L-shape

Fold upon itself

Anticodon

Nucleotide triplet forms complementary base-pair with mRNA codon, coding for amino acid

Aminoacyl tRNA synthetases

Activation of tRNA molecule with amino acid, carried out by enzymes

Enzymes catalyze tRNA to amino acid using ATP energy

Released from enzyme

Grows polypeptide chain on ribosome

Codon-Anticodon Pairing

Correct pairing of tRNA anticodon to mRNA codon

Base-pairing between mRNA codon and tRNA anticodon

Codon-anticodon pairing interactions

Wobble

First base (5') of codon, bind to last base (3')

Flexibility in base pairing between third nucleotide of codon, and tRNA anticodon

Initiation

Eukaryotes

5' cap of mRNA, scans until AUG start codon

Prokaryotes

Shine-Dalgarno sequences

Translation initiation complex assembles at 1+ ribosome binding sites

Polycistronic mRNA

Functionally related genes grouped together

Genes transcribed as single unit

Assembling Initiation Complex

Large & small subunits form functional ribosome when attached to mRNA molecule

Initiation factors bind to 5' cap of mRNA

Initiation factors bind to tRNA, charged with methionine

5'-3' until AUG

Functional Ribosomes

Methionine located in peptidyl (P) site

Charged tRNA enters & binds within aminoacyl (A) site

Peptide Bond Formation

Change in rRNA forms condensation reaction as peptide bond, transfers polypeptide chain into tRNA in A-site

Change in rRNA allowing fo peptidyl-transferase reaction

Ribosome translocates along mRNA

Enabled by binding of GTP-bound elongation factors

Cause deacylated tRNA to move from P-site to exit (E-site)

Termination

Ribosome reaches stop codon

mRNA sequence, GTP-bound release factors bind to A-site

Catalyze hydrolysis of terminal amino acid in polypeptide and tRNA in P-site

GTP hydrolysis dissociates translation complex, ribosomal subunits, tRNA

Genome to Protemone

Complexity

Proteome represents full number of proteins expressed by hereditary information in DNA

Genome

Single genes can encode multiple proteins

Reading, Interpreting, Processing Messages

Double membrane nucleus

Compartmentalization

Control in regulation of cellular processes

Mature mRNA exported out of nucleus

Cytosol

Free or ER bound ribosomes facilitate translation into polypeptides

Interpreting Signal

Cells Detect Changes in Environment

Stimuli resulting in cellular responses

Glucose Absorbed in Small Intestine

Absorbed Glucose Transported to Target Regions

Microvilli cells absorb glucose in intestinal tract

Sensori responses

Glucose absorbed into bloodstream

Micovilli cells

Associated with small blood vessels

Transported to blood vessles

Insulin

mRNA Processing & Protein Isoforms

Alternative Splicing

One pre-mRNA spliced at different junctions resulting in many different mature mRNA molecules

Different combination of transcribed exons

Exons may be excluded from splicing process

Removed

Produces isoforms

Mature mRNA from same pre-mRNA transcript

Regulate gene expression

Insulin

Termination

mRNA isoform translated into higher affinity insulin receptor in muscle cells

Lower blood glucose levels

Absorb glucose to meet high energy needs

Liver cells

Lower affinity insulin

High glucose stimulus detected in pancreas

Insulin is effector signal

Targets body's cells to absorb glucose from bloodstream

Increase glucose absorption

Antibiotics

Block growth & multiplication of microbes

Kill bacteria

Interfering with cell wall synthesis

Disrupting bacterial protein synthesis at ribosome

Inhibiting function of enzymes needed for DNA and RNA synthesis

Targets

tRNA binding sites

mRNA path

A, P, E sites

Tetracycline

Blocking A-site

Interfere with tRNA delivery

Streptomycin

Disrupting tRNA transfer

Prevent transfer of tRNA from A site to P site

Misreading

Brings inappropriate codon to binding site

Large ribosomal subuit

Oxazolidinones

Blocking large subunit

Translation cannot start

Chloramphenicol

Blocking peptide bond formation

Macrolides

Blocking peptide exit tunnel

Erythromycin

Translocation prevented

Move from A-site to P-site

Block P-site

Resistance

Inherited survival mechanism

Drug misuse

Misdiagnoses

Not taking full drug course

Chloramphenicol

rRNA mutations in large ribosomal subunit

Disrupts binding site

Methylation of rRNA prevents binding

Erythromycin

rRNA mutation at tunnel enterance

Add methyl group, block antibiotic binding