GENES & PROTEIN SYNTHESIS

central dogma

Francis Crick gave the name central dogma to the fundamental principle of molecular genetics, which states that genetic information flows from DNA to RNA to proteins

transcription

• the mechanism by which the information coded in DNA (genes) is transcribed into a complementary RNA copy

• occurs in the nucleus of eukaryotes

• unlike DNA, RNA can exit the nucleus and enter the cytosol

translation

• the assembly of amino acids into a polypeptide using the information encoded in the RNA

• takes place on the ribosomes in the cytosol

major types of RNA (3)

• messenger RNA (mRNA)

• transfer RNA (tRNA)

• ribosomal RNA (rRNA)

messenger RNA (mRNA)

• varies in length, depending on the gene that has been copied

• acts as the intermediary between DNA and the ribosomes

• is translated into protein by ribosomes

• is the RNA version of the gene encoded by RNA

transfer RNA (tRNA)

• functions as the delivery system of amino acids to ribosomes as they synthesize proteins

• very short, only 70-90 base pairs long

ribosomal RNA (rRNA)

• binds with proteins to form the ribosomes

• varies in length

transcription & translation in eukaryotes vs prokaryotes

• PROKARYOTES:

  • do not have nuclei

  • lack of compartmentalization allows translation of an mRNA to begin while transcription is still in progress

• EUKARYOTES:

  • the presence of a nuclear envelope separates transcription from translation

  • before eukaryotic RNA transcripts can leave the nucleus, they are modified in various ways to produce the final, functional mRNA

the genetic code

the specific amino acid coded for by particular DNA (or complementary RNA) bases is determined by the genetic code

Marshall Nirenberg

• determined the amino acid translation for each of the RNA codons

• found that the four bases in an mRNA must be used in combinations of at least three to provide the capacity to code for 20 amino acids (4³ = 64)

• he created artificial mRNA made entirely of uracil bases (UUU codons)

• when added to a test tube with the components for protein synthesis (amino acids, ribosomes), the mRNA directed the production of a polypeptide made only of phenylalanine (Phe)

• showed that codon UUU codes for amino acid phenylalanine

codon

• triplets of nucleotide bases are the smallest units of uniform length that can code for all amino acids; each three-letter combination is called a codon

• in translation, codons are read in the 5’ to 3’ direction along mRNA

• each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide

types of codons

• of the 64 codons, 61 specify amino acids; known as sense codons

• AUG specifies methionine; usually the first codon translated in any mRNA, so called a start codon

  • an enzyme may subsequently remove this amino acid from the chain

• three codons that do not specify amino acids (UAA, UAG, UGA) are stop codons; indicate the end of a polypeptide-encoding sentence

  • when a ribosome reaches a stop codon, polypeptide synthesis stops and the newly synthesized chain is released from the ribosome

redundancy in genetic code

• redundancy but no ambiguity

• e.g. GAA and GAG specify glutamic acid (redundancy) but neither specify any other amino acid (no ambiguity)

• also known as wobble hypothesis

• presence of redundancy allows the third base in the codon to change (wobble) while still allowing the codon to code for the same amino acid

important characteristics of genetic code (3)

• redundancy: more than one codon can code for the same amino acid

• continuity: code is read as a series of three-letter codons without spaces, punctuation, overlap

• universality: almost all organisms build proteins with the same genetic code

stages of transcription/translation

• initiation

• elongation

• termination

transcription initiation

• transcription begins when the enzyme RNA polymerase binds to the DNA and unwinds it near the beginning of a gene

• binding occurs at a promoter

promoter

• a specialized nucleotide sequence on one strand of DNA

• located just upstream from the start of the gene

• allows the binding of RNA polymerase

• different types are similar enough that RNA polymerases and helper proteins are able to recognize it

initiation in transcription vs replication

• RNA polymerases do not need to add the first nucleotide onto a pre-existing primer

• once RNA polymerase is positioned at the promoter, it causes the DNA double helix to partially unwind without the help of helicase

• RNA polymerase uses energy from binding and conformational changes (not ATP) to open 12-14 base pairs of DNA to form the transcription bubble

transcription initiation in prokaryotes

• key element of the promoter is a TATAAT sequence upstream the start point

  • section of DNA with high percentage of T and A bases, recognized by RNA polymerase

• once RNA polymerase binds to the promoter, a conformational change occurs, locking DNA in place and causing breaking of hydrogen bonds in TATAAT sequence

• part of the gene that is to be transcribed into RNA is called transcription unit

reason for TATA box and TATAAT sequence

• adenine and thymine share only two hydrogen bonds

• since less energy is needed to break two bonds, RNA polymerase expends less energy opening up the DNA helix

transcription initiation in eukaryotes

• a collection of proteins called transcription factors, one recognizing the TATA box, must bind to the DNA before RNA polymerase II can bind to the promoter in the correct position & orientation

• whole complex of transcription factors and RNA polymerase II binding to promoter is called transcription initiation complex

transcription elongation

• RNA polymerase builds RNA molecules in the 5’ to 3’ direction, using 3’ to 5’ DNA strand as template strand

• as RNA polymerase moves along DNA, it untwists the helix by disrupting hydrogen bonds

• as RNA synthesis advances, the new RNA molecule peels away from its DNA template, and the DNA double helix reanneals

• the reforming of the DNA double helix pushes the RNA out further, with a small bubble remaining open

coding strand

• in transcription, the opposite strand of DNA (strand not being copied)

• contains the same base-pair sequence as the new RNA molecule, except for substitution of T for U

multiple RNA polymerase molecules during transcription

• once an RNA polymerase molecule has started transcription and progressed past the beginning of a gene, another molecule of RNA polymerase may start producing another RNA molecule if there is room at the promoter

• allows the cell to make encoded protein in large amounts

• e.g. a single red blood cell contains 375 million hemoglobin molecules

dystrophin

• largest human gene encodes the protein dystrophin

• missing or non-functional in the disease muscular atrophy

• gene is 2.5 million nucleotides in length and takes over 16 hours to produce a single mRNA transcript

transcription termination

transcription ends when RNA polymerase recognizes a termination sequence

transcription termination in prokaryotes

• one termination mechanism involves a protein binding to the mRNA and stopping transcription

• the transcribed terminator (RNA sequence) functions as the terminal signal, causing the RNA polymerase to detach from the DNA and release the transcript

• transcript requires no further modification before translation

transcription termination in eukaryotes

• RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal sequence, which specifies a signal (AAUAAA) in pre-mRNA

• nuclear proteins bind to the polyuracil site and stop transcription by cutting the RNA transcript free from the polymerase

• newly synthesized RNA dissociates from DNA template strand

• transcription ceases, and RNA polymerase is free to bind to another promoter region and transcribe another gene

post-transcriptional modifications (2)

• capping & tailing

• splicing

poly(A) tail

• the addition of a chain of 50-250 adenine nucleotides, one nucleotide at a time, to the 3’ end

• done by an enzyme called called poly-A polymerase

• process is called polyadenylation

• the adenine chain is called the poly(A) tail and enables mRNA to be translated efficiently and protects it from attack by RNA-digesting enzymes in the cytosol

5’ cap

• modifications are made at the beginning of the pre-mRNA transcript, where a 5’ cap, consisting of seven Gs (modified G nucleotides) is added by a different enzyme complex

• 5’ cap functions as the initial attachment site for mRNAs to ribosomes, to allow for translation

capping & tailing functions (3)

• they facilitate the export of mature mRNA from the nucleus

• they help protect the mRNA from degradation by hydrolytic enzymes

• they help ribosomes attach to the 5’ end of the mRNA once the mRNA reaches the cytoplasm

untranslated regions (UTRs)

• at the 5’ and 3’ ends of the mRNA

• parts of the mRNA that will not be translated into protein

• function to allow ribosomes to bind to during translation

introns & exons

• eukaryotic DNA is composed of coding regions (exons) and non-coding regions (introns)

• introns do not code for part of the protein and if left would alter the sequence of amino acids

• would result in additional amino acids and a protein that would not fold/function properly

• presence of introns may facilitate evolution of new and potentially beneficial proteins as a result of exon shuffling

mRNA splicing

• removes introns from pre-mRNA molecules and joins exons together, forming an mRNA molecule with a continuous coding sequence

• spliceosome formed from snRNPs; cleaves the intron at its beginning

• intron folds back and bonds to itself; it is released and rapidly degraded

• spliceosome joins together two exons

spliceosome

• complex formed between pre-mRNA

• made from five small ribonucleoproteins (snRNPs)

• snRNPs bind to the intron by recognizing its boundary sequences, forming complementary base pairs, and loop the intron out, bringing the two exons close together

snRNP purposes

• participate in spliceosome assembly and splice site recognition

• also catalyze splicing reaction

alternative splicing

• exons may be joined in different combinations to produce different mRNAs from a single DNA gene sequence

• alternative splicing increases the number and variety of proteins encoded by a single gene

how alternative splicing is done

• done by including or skipping certain exons, using different splice sites (start and end sites), retaining an intron

• splicing can be altered by RNA polymerase speed (forgetting exons), cell signals for stress, hormones, developmental cues, enhancers, silencers

• different tissues might splice the same gene differently; e.g. muscle cells produce different isoforms of tropinin T gene in skeletal vs cardiac muscle

transcription in eukaryotes vs prokaryotes (7)

• location: throughout the cell; in the nucleus

• enzymes: single type of RNA polymerase; different DNA polymerases used to transcribe genes that encode protein and genes that do not

• elongation: bases added quickly (15-20/s); bases added slowly (5-8/s)

• promoters: less complex, more complex

• termination: protein binds to mRNA and cleaves it or mRNA binds to itself; nuclear proteins bind to polyuracil site

• introns & exons: no introns; both introns and exons

• product: mRNA ready to be translated; results in pre-mRNA

function of tRNA

• to transfer an amino acid from the cytoplasm to a growing polypeptide in a ribosome

• each tRNA molecule is used repeatedly, picking up its designated amino acid in the cytoplasm, depositing the amino acid onto a polypeptide chain at the ribosome, and then leaving the ribosome to pick up another of the same amino acid

tRNA structure

• regions that base pair with themselves, winding into four double-helical segments to form a cloverleaf

• 5’ and 3’ ends are both located near one end

• at one tip of one double-helical segment is an anticodon

• at 3’ end is a region that carries the amino acid that corresponds to the anticodon

• e.g. serine pairs with codon 5’-AGU-3’ in mRNA, anticodon of tRNA is 3’-UCA-5’

anticodon

3-nucleotide segment that pairs wth codon in mRNA

wobble hypothesis

• the complete set of 61 codons can be read by fewer than 61 distinct tRNAs

• pairing of the anticodon with first two nucleotides of the codon is always precise, but most anticodons have flexibility in pairing with the third nucleotide of the codon

aminoacylation

• the process of adding an amino acid to a tRNA

• “charging” the tRNA

• the correct matching up of tRNA and its amino acid is carried out by a family of enzymes called aminoacyl-tRNA synthetases (aaRS)

• the finished product, a tRNA linked to its correct amino acid, is called an aminoacyl-tRNA

• catalyzed by 20 different aaRS enzymes, one for each of the 20 amino acids

aminoacylation process

• one synthetase is able to bind to all the different tRNAs for its particular amino acid

• the active site of each type of aaRS fits only a specific combination of amino acid and tRNA

• the aaRS catalyzes the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP

energy that allows formation of peptide bond in translation

energy in the aminoacyl-tRNA drives the formation of the peptide bond

ribosome structure

• consists of a large and a small subunit, each made up of proteins and ribosomal RNAs

• about 1/3 mass is made of proteins; the rest consists of 3 (bacteria) or 4 (eukaryotes) rRNA molecules

ribosome function in translation

carry out protein synthesis by translating mRNA into chains of amino acids

ribosome assembly

• subunits made in the nucleolus

• completed ribosomal subunits are exported via nuclear pores to cytoplasm

• subunits join to form a functional ribosome only when attached to an mRNA molecule

ribosomal binding sites

• A site (aminoacyl-tRNA binding site): holds the aminoacyl-tRNA carrying the next amino acid to be added to the chain

• P site (peptidyl-tRNA binding site): where the tRNA carrying the growing polypeptide chain is bound

• E site (exit site): discharged tRNAs leave the ribosome

translation initiation

• the first components to associate with each other are mRNA, a specific initiator aminoacyl-tRNA (bearing the first amino acid, methionine), and a small ribosomal unit

• components are brought together by proteins called initiation factors

• large ribosomal subunit binds to complete the ribosome

• at the end of initiation, the initiator met-tRNA is in the P site

• tRNA also helps to keep the mRNA in place

• amino acids added to at the C-terminus of a growing chain

translation initiation: prokaryotes

the ribosomal subunit binds the mRNA at a specific RNA sequence, just upstream of the AUG start codon

translation initiation: eukaryotes

• the initiator met-tRNA forms a complex with the small ribosomal subunit

• the complex binds to the mRNA at the 5’ cap and then moves along the mRNA (scanning) until it reaches the first AUG (start) codon

• met-tRNA hydrogen bonds to the start codon

energy expended in translation

obtained by hydrolysis of a guanosine triphosphate molecule to form the complex

translation elongation steps (3)

1. codon recognition

2. peptide bond formation

3. translocation

translation elongation: codon recognition

• the anticodon of an incoming aminoacyl-tRNA base pair with the complementary mRNA codon in the A site

• however, met-tRNA is first bound to the P site

• requires hydrolysis of one molecule of GTP, which increases accuracy and efficiency

translation elongation: peptide bond formation

• the second tRNA, with an anticodon and amino acid (AA₂), binds to the codon in the A site of the ribosome

• the amino acid (Met) is cleaved from the tRNA in the P site and forms a peptide bond with the amino acid (AA₂) in the A site

• the new polypeptide chain is attached to the tRNA in the A site and an empty tRNA remains at the P site

• bond formation is catalyzed by peptidyl transferase, a ribosomal enzyme

translation elongation: translocation

• the ribosome translates the tRNA in the A site to the P site

• at the same time, the empty tRNA in the P site is moved to the E site, where it is released

• one more GTP hydrolyzed to provide energy for the translocation step

amount of aminoacyl tRNAs present in translation

many are present, but only the one with the appropriate anticodon will bind and allow the cycle to progress

translation termination

• elongation ends when the A site of a ribosome arrives at one of the stop codons on the mRNA

• when a stop codon appears on the A site, a release factor binds at this site instead of an aminoacyl-tRNA

• release factor causes the addition of water molecule instead of an amino acid to the polypeptide chain; hydrolyzes bond between completed polypeptide and tRNA in the P site

• as no amino acid is present at the A site, the polypeptide chain detaches from the ribosome, leaving through the exit tunnel of the ribosomal large subunit

• ribosomal subunits separate and detach from mRNA; empty tRNA and release factor are released

release factor

protein shaped like an aminoacyl-tRNA

protein functionality after translation

• polypeptide is still not function; exists in an inactive state

• the polypeptide chain must be folded into the correct formation, which is done by multiple processing reactions to activate the polypeptide

• proteins composed of multiple chains; polypeptides produced from a number of separate translation events are processed and then assembled together to form a single functioning protein

polysome

• a complex that is formed when multiple ribosomes attach to the same mRNA molecule in order to facilitate rapid translation

• once a ribosome is far enough past the start codon, a second ribosome can attach to the mRNA, resulting in a number of ribosomes trailing along the mRNA

point mutations

mutations in a single nucleotide in a gene

types of point mutations (3)

• substitution of one base for another

• the insertion or deletion of a single pair

• the inversion of two adjoining base pairs

single nucleotide polymorphisms (SNPs)

• differences in the DNA of individuals within a population that are caused by point mutations

• effect can range from being positive, to having no effect, to being severe

types of small-scale mutations (4)

• missense mutation

• nonsense mutation

• silent mutation

• frameshift mutation (insertion & deletion)

missense mutation

• a change of a single or group of base pairs; results in the code for a different amino acid

• synthesized protein will have a different sequence and structure, may be nonfunctional or function differently

• can be beneficial if it creates a new, desirable effect

nonsense mutation

• change of a single or group of base pairs results in a premature stop code in the gene

• polypeptide is cut short and, most likely, will be unable to function

silent mutation

• change in one or more base pairs does not affect the functioning of the gene

• mutated DNA sequence codes for the same amino acid as the non-mutated sequence, and the resulting protein is not altered

frameshift mutation

• one or more nucleotides are inserted/deleted from a DNA sequence, causing the reading frame of codons to shift in one direction or the other

• results in multiple missense/nonsense effects

• every amino acid coded for after this mutation is affected

lactose metabolism

• lactose metabolism begins with its hydrolysis into its component monosaccharides (glucose + galactose)

• reaction catalyzed by the enzyme ß-galactosidase

lac operon

• a cluster of three genes that code for the proteins involved in the metabolism of lactose

• consists of:

  • a promoter (site where transcription begins)

  • an operator (controls access of RNA polymerase to genes)

  • coding regions for various enzymes that actually metabolize lactose

• includes three structural genes for lactose metabolism: lacZ, lacY, lacA

lac operon structural genes (3)

• lacZ: codes for ß-galactosidase, which hydrolyzes lactose to glucose and galactose

• lacY: codes for permease, the membrane protein that transports lactose into the cell

• lacA: codes for transacetylase, an enzyme that detoxifies other molecules entering the cell via permease

lac operon repressor protein

• upstream from the operon is a gene that codes for an allosteric repressor protein

• protein takes cues from lactose concentration in the cell and regulates the production of the lactose-metabolizing proteins

• repressor protein can switch off the lac operon by binding to the lac operator

• lacI protein or lac repressor

• genes that code for the lac repressor are always transcribed, so lac repressor is always present within a cell

lac repressor when lactose is absent

• the lac repressor is active and binds to the operator

• genes of the lac operon are silenced

• this keeps RNA polymerase from binding to the promoter region and stops the lactose-metabolizing enzymes from being synthesized

lac repressor when lactose is present

• allolactose binds to the lac repressor and alters its shape so the repressor can no longer bind to the operator

• inactive lac repressor is unable to bind to the operator and block transcription

• now RNA polymerase can bind to the promoter region, and transcription of lac genes begins

allolactose

• allolactose (isomer of lactose) binds onto a site on the lac repressor, rendering it inactive; called an inducer

• allolactose formed when ß-galactosidase, instead of breaking down lactose, rearranges it into allolactose

inducible vs repressible operon

• lac operon is an inducible operon because the inducer inactivates the repressor and allows the gene to be transcribed by binding allosterically to a regulatory protein (lac repressor)

  • as concentration of lactose increases/decreases, so does the transcription of lac genes

  • helps cell conserve energy

• enzymes for tryptophan synthesis are repressible because transcription is repressed when tryptophan binds allosterically to regulatory protein (trp repressor)

tryptophan synthesis

• E. coli synthesizes amino acid tryptophan from a precursor molecule in the three-step pathway

• each reaction in the pathway is catalyzed by a specific enzyme, and the five genes that code for subunits of these enzymes are clustered together on the bacterial chromosome

trp operon

• made of operator, promoter, and the genes they control (DNA required for enzyme production for tryptophan pathway)

• also a gene that codes for a trp repressor protein; it is always synthesized, but is only activated in the presence of tryptophan

advantage of grouping genes of related function into one transcription unit

a single “on-off switch” can control the whole cluster of functionally related genes

trp operon repressor protein gene

• a repressor protein is encoded by a regulatory gene called trpR

• trpR is located some distance from the trp operon and has its own promoter

• trp repressor is an allosteric protein and is either active or inactive

  • synthesized in the inactive form, which has little affinity for trp operator

  • only when tryptophan binds to trp repressor at allosteric site does the repressor change to the active form to turn the operon “off”

  • reversible

trpR when tryptophan is absent

• the repressor protein is in an inactive state and does not bind to the operator

• RNA polymerase is able to bind to the promoter region, and the transcription of genes responsible for the biosynthesis of tryptophan can proceed

trpR when tryptophan is present

• the cell can conserve energy by using available tryptophan and stopping the transcription of the genes that code for enzymes involved in tryptophan biosynthesis

• tryptophan (signal molecule) activates the repressor protein, which binds to the operator to stop transcription

  • when a signal molecule acts this way, it is called a corepressor, serving to repress the expression of a set of genes

genetic engineering

the intentional alteration of a genome by substituting or introducing new genetic material into the genome

bacteria for genetic engineering

bacteria are versatile tools for genetic engineers because they reproduce quickly and often, are relatively inexpensive to maintain, and contain plasmids

plasmids

small circular pieces of DNA that replicate independently of the bacteria’s chromosome

restriction enzymes

• first step in genetic recombination is to isolate/cut out a DNA fragment that contains the desired gene

• scientists use restriction enzymes (endonucleases) which occur naturally in prokaryotic cells

• restriction enzymes protect the bacterial cell by cleaving foreign DNA from other organisms or phages, thus restricting the replication of infecting viruses

• DNA of a bacterial cell is protected from the cell’s own restriction enzymes through DNA methylation

how restriction enzymes work

• each restriction enzyme recognizes a specific sequence of nucleotides on a DNA strand (recognition site for a particular enzyme)

• when the restriction enzyme cuts the DNA molecule, the pieces it creates are known as restriction fragments

• most commonly used restriction enzymes recognize sequences containing 4-8 nucleotide pairs

  • probability of finding a particular four-pair sequence is one in 4⁴ (0.4%)

characteristics of restriction enzymes (2)

• sequence specificity: cuts are specific and predictable; the same enzyme will cut a particular strand of DNA the same way, producing identical sets of restriction fragments

  • each restriction enzyme cuts at only one recognition site and in only one direction

• staggered cuts: most restriction enzymes leave a few unpaired nucleotides on a single strand at each end of the restriction fragment (sticky ends), which can form base pairs with other single-stranded regions with a complementary sequence

restriction site recognition (2)

• hydrogen bonding with specific bases in the major and minor grooves of the DNA

• shape recognition: 3D structure of the restriction site fits perfectly into the enzyme’s active site

restriction enzyme naming

• named after cell of origin, plus Roman numeral if more than one restriction enzyme has been isolated from the species

• e.g. EcoRI (“eco-R-one”) was the first restriction enzyme isolated from E. coli

EcoRI restriction enzyme

• EcoRI cuts the phosphodiester bond in the DNA backbone between the G and the A

• DNA sites are palindromic

• another EcoRI enzyme makes the same cut in the complementary DNA strand

• this leaves only a small number of the hydrogen bonds holding the DNA together, allowing the DNA molecule to be easily separated, resulting in complementary “sticky” ends

sticky & blunt ends

• if cuts are made straight across the strand, blunt ends are created

• if cuts are made in a zig zag, sticky ends are created

• e.g. EcoRI produces sticky ends, SmaI produces blunt ends

• molecular biologists prefer to work with restriction enzymes that produce sticky ends because the DNA fragments created are easier to join to any other DNA strand that has been cut by the same enzyme

DNA ligase

• enzyme that is used to join cut strands of DNA by forming phosphodiester bonds between their backbones

• for successful joining, the DNA fragments must be complementary, which is achieved by using the same restriction enzyme

• works best with sticky ends of DNA, but a second form, T4 DNA ligase, works well with blunt ends

how DNA ligase seals gaps for restriction fragments

• hydrogen bonds form between the complementary bases, but this is not stable; DNA is not fully linked until phosphodiester bonds form between the backbones

• DNA ligase stabilizes the connection by catalyzing a dehydration reaction that creates covalent bonds, releasing water in the process

plasmid replication + DNA makeup

• plasmids replicate independently of the chromosomal DNA

• often contain genes that code for specific proteins, such as proteins that provide resistance to antbiotics or protect from toxicity of heavy metals

competent cell

a cell that is able to take up foreign DNA (plasmid) from its surroundings, such as an E. coli cell