ch7 - the blueprint of life

DNA

characteristics of each cell dictated by info contained within dna

• def: the blueprint of life; hereditary material that is passed onto offspring

• contains all info required to make the organism and give the organism all its abilities

genome

• complete set of genetic info [genotype = specific combination of alleles (gene variants) an individual possesses], all the genes they contain

• genes are composed of sections of dna in all living organisms

  • chromosomes are made of dna

• functional unit is the gene

  • genes code for products (protein, RNA)

phenotype

physical characteristics or enzymes resulting from the expression of genes

to multiply, cells must carry out:

dna replication

gene expression (transcription and translation)

• info flow from dna → rna → protein

  • known as the central dogma of molecular bio

characteristics of dna

made of deoxyribonucleotides

DNA: double stranded molecule

• strands are complementary due to the specific pairing of bases

  • AT, CG

• strands held together by hydrogen bonds

  • melting or denaturing - separate strands

  • there are 2 hydrogen bonds between A and T

  • 3 hydrogen bonds between C and G

  • the more Gs and Cs they have, the more hydrogen bonds they have, the harder it is to split it, so they have a high boiling point, and then the strands get separate

• strands are antiparallel

  • 5’ phosphate to 3’ hydroxyl and 3’ hydroxyl to 5’ phosphate

characteristics of rna

made of ribonucleotides

• Uracil replaces Thymine in RNA, U vs T

shorter, single stranded molecule

3 types of RNA: each with a different function

• protein encoding gene: section of dna providing instructions for making a protein → mRNA → translation → protein

• rRNA gene: section of DNA, structural RNA and makes up the ribosome (protein making) → ribosomal RNA

• tRNA gene: section of DNA → tRNA, RNA molecules that will bring the amino acid to the ribosome during process of translation or protein synthesis

DNA replication

duplication of genome, the bacteria, the chromosome

• its circular

begins at the origin of replication

• melt/break apart/unwinds the double stranded DNA

• each original strand is used as a template to make the new complementary strand

bi-directional synthesis of DNA

• replication will keep going in both directions from the origin until it meets

• creates 2 replication forks

• forks continue moving around the circular chromosome until they meet

semiconservative

• 2 DNA molecules, each with an old and new strand

process of dna replication

dna replication uses dna polymerase

IMPORTANT:

• reads dna template 3’ to 5’

• dna polymerase always synthesizes in the 5’ to 3’ direction

can only add nucleotides to an existing molecule, not initiate

• helicase unzips 2 strands of DNA

• dna polymerase requires RNA primers at the origin of replication to start synthesis

• the first thing is the synthesis of RNA primer

• 5’ — (3’ to 5’) — 3’

  • () is rna molecule, primer

  • the RNA primer gives dna polymerase a starting point

  • it can add nucleotides to 3’ -OH end of the strand only

  • DNA polymerase adds DNA nucleotides

process of dna replication - leading and lagging

dna strands are antiparallel → grow in opposite directions

• leading strand: synthesize continuously in 5’ to 3’ direction (5→3→5→3 etc)

  • synthesizing on the 5’ to 3’ strand towards the replication fork

• lagging strand produces Okazaki fragments: synthesizing in small fragments, discontinuously

  • synthesizing on the 5’ to 3’ strand away from the replication fork in Okazaki fragments

  • it moves down and puts a RNA primer and synthesizes a small piece

  • move down again and synthesize again

  • makes RNA primer

  • DNA polymerase adds DNA to the primer making a small DNA section

  • replication fork opens up more

  • makes another RNA primer

  • repeat

THEN:

• DNA ligase seals the gaps between Okazaki fragments by forming a covalent bond between them

• as DNA polymerase adds nucleotides to the 3’ end of one Okazaki fragment, it encounters the 5’ end of another

• a different type of DNA polymerase then removes the RNA primer nucleotides and simultaneously replaces with deoxynucleotides

gene expression in bacteria - transcription

synthesis of a complementary strand of mRNA from a portion of the DNA by RNA polymerase

important: RNA polymerase binds to promoter

• the promoter determines where transcription starts and which DNA strand the RNA polymerase will use as a template

• can run in either direction

• RNA polymerase reads the template strand 3’ → 5’

• RNA polymerase synthesizes the new RNA strand 5’ → 3’

stops at terminator

mRNA

• carries info from DNA to ribosome

3 steps of transcription process: Initiation, Elongation, Termination

gene expression in bacteria - transcription/ initiation

• RNA polymerase binds to the promoter with the help of a sigma factor and melts/unwinds a short section of DNA double helix

  • eukaryotes use transcription factors not sigma factors

• function of sigma factor is to guide RNA polymerase to promoter

• RNA polymerase chooses one DNA strand as the template

• can initiate without RNA primer, RNA polymerase doesn’t require it

• RNA synthesis begins

gene expression in bacteria - transcription/ elongation

• sigma factor dissociates from RNA polymerase, leaving the core enzyme of RNA polymerase to complete transcription

• RNA is synthesized in the 5’ to 3’ direction

  • read in 3’ → 5’

• the enzyme adds nucleotides to the 3’ end of the growing RNA strand

• new RNA strand is complementary and antiparallel

• itll continue on and on until it reaches the terminator

• eukaryotic doesn’t require sigma factor, only bacteria

gene expression in bacteria - transcription/ termination

• when RNA polymerase encounters a termination, transcription stops and it falls off the template releasing the newly synthesized RNA from the DNA

gene expression in bacteria - translation

process of decoding the information in mRNA → protein

also involves

• ribosomal RNA (rRNA)

  • forms ribosome - “translational machine”

• transfer RNA (tRNA)

  • brings correct amino acid to ribosome

  • carries anticodon

mRNA read three nucleotides at a time (3 n = codon)

most codons specify a particular amino acid according to the genetic code

the genetic code is redundant (degenerate)

64 possible codons

• 61 codons for amino acids, 3 are stop/nonsense codons

• AUG

  • start codon for most protein

  • also, codes for the amino acid methionine/met

• UAA, UAG, UGA

  • stop codon

specific nucleotide sequences define different regions on the mRNA

• ribosome binding site - where the ribosome binds to begin translation

• start and stop codons mark the beginning and end of the region to be translated

anticodon

• anticodon on tRNA binds to the complementary codon on mRNA through hydrogen bonds

• this allows tRNA to recognize and match the correct codon on the mRNA

• before entering the ribosome, the tRNA is attached to its specific amino acid by an enzyme called aminoacyl-tRNA synthetase

• the enzyme ensures that the correct amino acid is attached to the correct tRNA

• therefore, when the anticodon matches the mRNA codon, the tRNA brings the correct amino acid to the ribosome

translation process - initiation

initiating tRNA brings formylmethionine (f-Met) to the P-site of the ribosome

• tRNA anticodon pairs with start codon

• first tRNA carries 1st amino acid to the start codon AUG

• only in bacteria its f-Met instead of methionine

• translational machinery ready after assembling

• A-site: aminoacyl

• P-site: peptidyl

• E-site: exit

  • first tRNA/start codon will be in P-site

translation process - elongation

• synthesis of a polypeptide to make a longer protein

• another tRNA carries the amino acid that matches the codon into the A-site

  • recognizes codon in empty A-site

  • brings in next correct amino acid and fills that site

• ribosome catalyzes the joining of the amino acid carried by the tRNA in the P-site to the one carried by the tRNA in the A-site

  • a ribozyme creates a peptide bond between 2 amino acids

• the ribosome advances a distance of one codon (3 nucleotides)

  • the tRNA that occupied the P-site exits through the E-site

  • the tRNA that was in the A-site moves into the P-site

  • a tRNA that recognizes the next codon quickly fills the empty A-site

    • example: the next one is Tyrosine and tRNA carries it into the A-site

    • ribosome forms a peptide bond between the growing dipeptide of Pro and f-Met, and it will attach to Tyr

• mRNA advances in 5’ to 3’ direction through ribosome

  • initiating tRNA exits through the E-site

  • tRNA recognizing next codon in A-site attaches

• peptide bond forms between amino acids

  • ribosome advances one codon on mRNA

  • empty tRNA exits E-site, new tRNA occupies A-site

• process repeats until we get to the end of the mRNA which is its stop codon, and a release factor binds to it causing the completed polypeptide to be released

translation process - termination

• elongation stops when ribosome reaches stop codon

  • when the stop codon enters in the A site

    • a release factor recognizes the stop codon bcuz no tRNA molecules recognize or can bind to it.

    • the release factor triggers release of the completed polypeptide and termination of translation

• ribosome falls off mRNA

  • components disassemble releasing the newly formed polypeptide

  • dissociates into subunits (30S) and (50S)

  • mRNA and completed polypeptide released

  • then the proteins fold after synthesis

• after translation

  • newly synthesized polypeptide protein folds into its function shape

  • may undergo modifications depending on the protein

simultaneous transcription and translation

only in bacteria both processes can occur at the same time

• IMP- why only bacteria: 2 reasons

  • bacteria is a prokaryote, so doesnt have a nucleus

  • doesn’t occur in eukaryotes bcuz in eukaryotic transcription it occurs in the nucleus and translation occurs in the cytoplasm in different regions

  • Eukaryotic mRNA/DNA has introns and exons (non coding vs coding)

    • when mRNA is made it has both introns and exons

    • all introns have to be removed and exons spliced together → mature mRNA leaves the nucleus and enters the cytoplasm where it will be translated by a ribosome

  • Insulin/cDNA example:

  • protein made from RNA produces correct insulin protein

  • scientists can convert mature human mRNA through splicing and removal of introns and turn it into cDNA (complementary DNA)

  • introduces the cDNA into the bacteria so the bacteria can produce the desired human protein

  • and this is what we pump in bacteria to produce the correct protein bcuz the cDNA from mRNA doesnt have any introns

translation of the mRNA begins while the mRNA is still being synthesized

• polyribosomes: multiple ribosomes translating the same mRNA

promoter is a DNA sequence where RNA polymerase binds to start transcription

• going to use blue in this example because thats where the promoter is for 3’ → 5’

as soon as the mRNA is done being transcribed in ribosome binding site, the ribosomes hop on and start translating

bacteria can adjust quickly because they can do both quickly at the same time in a minute or two

• they alr have the proteins they need to be functional and can adjust to any environment

• beginning of mRNA is where the ribosome binding site is and where the start codon for translation to begin is

• as we go down mRNA it goes longer and longer

bacterial gene regulation

control of transcription/translation/gene regulation

allow bacteria to

• conserve energy

  • bacteria are very conservative - they will not waste energy making products they dont need

    • will rather put that energy in growth

• respond/adapt to changing environments by altering the level of gene expression

  • changing which genes they express

  • may turn some genes off and others on to make new proteins depending on the environment

constitutive genes

constantly expressed, enzymes produced

• proteins made all the time bcuz they’re needed to maintain the life and basic ongoing functions of the cell

• ex: enzymes of glycolysis need to be available regularly cuz theyre needed to break down glucose

  • need it all the time bcuz glucose is an important energy source for cells

regulated genes

can be turned on/off; only made when needed

inducible genes

• we can induce it meaning we can turn it on when we need them

• not regularly expressed

• turned on only under certain conditions (induction)

  • B-galactosidase - only when lactose is present

  • lactose → allolactose (inducer)

    • inducer turns on inducible genes

• mechanisms can turn on transcription for as long as needed

  • ex: when the substrate for an encoded enzyme is present

repressible genes

• repressed meaning it can be turned off

• routinely expressed, can be turned off when not needed

• can turn off transcription for as long as necessary

  • ex: when the product of an encoded enzyme is in sufficient quantity

• generally involved in biosynthesis

  • amino acids (tryptophan (trp) )

    • genes in trp are repressible

    • bacteria are making trp and we suddenly give it a lot of trp

    • if theres a lot in the surroundings, it wil use a little bit of energy to transport it in the cell

    • genes involved in this are routinely expressed and it will turn those genes off with a repressor if theres suddenly a lot

• controlled by a repressor that blocks transcription when genes are no longer needed → repression

levels of regulation

control of transcription

• induction and repression

• so mRNA is not made

• most efficient way of saving energy

control of translation

• rapid degradation of mRNA transcripts so that it doesnt get translated

protein activity

• even if its made we can control the enzyme activity (protein function)

  • feedback inhibition

    • overabundance of end-product (enzymes) inhibits enzymes that makes it

    • will stop the enzyme function

    • amino acid Threonine converted by the steps to make Isoleucine

      • when theres a lot of Isoleucine, it will bind to and inhibit the function of the first enzyme in the pathway

      • when it does that itll stop the function of the enzyme and the next intermediate will not be made

      • therefore it cant be converted to the next and the next, therefore Isoleucine will not be made

      • a lot of Isoleucine around so cell doesnt need it