RNA and the Genetic Code

The Genetic Code

• Genetic code is used to translate genetic information into proteins
• Proteins are what helps the organism develop and perform the necessary functions of life.
• Central Dogma of Molecular Biology: the major steps involved in the transfer of genetic information
  • DNA -> RNA -> Protein
• Gene: a unit of DNA that encodes a specific protein or RNA molecule
  • Gene can then be expressed through transcription and translation
• Messenger RNA is synthesized in the 5’ to 3’ direction and is complementary and antiparallel to the DNA template strand
  • Ribosomes translate the mRNA in the 5’ to 3’ direction as it synthesizes the protein from the amino terminus (N-terminus) to the carboxy terminus (C-terminus)

Types of RNA

Messenger RNA (mRNA)

• Carries the information that specifies the amino acid sequence of the protein to the ribosome
• Transcribed from DNA template plates by RNA polymerase enzymes that are in the nucleus of cells.
• mRNA can go through many transformations modification after transcription and before it is released from the nucleus.
• The only type of RNA that contains information that can be translated to a protein.
  • Read in three-nucleotide segments, called codons.
• Monocistronic: in eukaryotes, each mRNA molecule translated into only one protein product.
• Polycistronic: in prokaryotes, starting the process of translation at different locations in the mRNA can result in different proteins.

Transfer RNA (tRNA)

• Responsible for converting the language of nucleic acids to the language of amino acids and peptides.
• Each tRNA molecule contains a folded strand of RNA that includes a three-nucleotide anticodon.
• While in the ribosome, an anticodon is responsible for recognizing and pairing with the appropriate codon on an mRNA
• There are 20 amino acids in eukaryotic proteins and each has to be represented by at least one codon.
• Amino acids are connected to a specific tRNA molecule. Molecule is said to be charged or activated with an amino acid.
• Mature tRNA can be found in the cytoplasm
• Each amino acid is activated by different aminoacyl-tRNA synthetase
  • Requires two high energy bonds from ATP. Which means that the attachment of the amino acid is an energy-rich bond.
  • Transfers the activated amino acid to the 3’ end of the correct tRNA
    • Amino acids are bound to a CCA nucleotide sequence
  • This high energy bond is used to supply the energy needed to create a peptide bond during translation

Ribosomal RNA (rRNA)

• Synthesized in the nucleolus and functions as an integral part for the ribosome during protein assembly
• Many function as ribozymes: enzymes made of RNA molecules instead of peptides
  • Helps catalyze the formation of peptide bonds
  • Also important in splicing out its own introns (opposite of DNA encoding portions called exons) within the nucleus.

Codons

• Each codon consists of three bases and they are translated into an amino acid
• 64 codons in total and are written in 5’ to 3’ direction
  • 61 codons encode for 20 amino acids while 3 codons encode the termination of translation. (UAA, UAG, UGA are the universal stop codes)
• During translation the codon of an mRNA is recognized by a complementary anticodon on the tRNA
• Codon and anti must be antiparallel. E.g. – anticodon: 5’-GAU-3’; Codon: 5’-AUC-3’
• Every eukaryotic proteins starts with methionine. This is considered the start codon (AUG) for translation of the mRNA into protein
• tRNA molecules that recognize the stop codons (UGA, UAA, and UAG) are not charged, and they encode the release of the protein from the ribosome.

Mutations

Degeneracy and Wobble

• Genetic code is degenerate since more than one codon can specify a single amino acid
• All amino acids except for methionine and tryptophan are encoded by multiple codons
• For the most part, the first two bases in a codon that encode the same amino acid are the same.
  • Third base is variable and called the wobble position.
• Wobble position is designed to protect against mutations in the coding region of DNA
  • Mutation in the wobble position tend to be called silent or degenerate, which insinuates that the mutation has no effect on the expression of the amino acid.

Missense and Nonsense Mutations

• Point mutations: mutation only affects one nucleotide in a codon. If in a wobble position, mutation is not expressed, otherwise the mutation can be expressed.
• Known as expressed mutations since these mutations can affect the primary amino acid sequence. Falls into two categories:
  • Missense Mutations: mutation where one amino acid substitutes for another
  • Nonsense mutation: where the new codon encodes for a premature stop codon (also known as truncation mutation)

Frameshift Mutations

• Reading Frame: three nucleotides of a codon
• Frameshift mutations: when some number of nucleotides is added or deleted from the mRNA sequence.
• Usually results in an amino acid sequence change or premature truncation of the protein
• Generally regarded as more serious than point mutations

Transcription

• DNA contains info to code the gene. Actual gene making is done in the cytoplasm
• DNA cannot leave the nucleus, which is why it uses RNA to transmit the information
• Transcription: creation of mRNA from a DNA template.

Mechanism of Transcription

• Transcription produces a copy of only one of the two strands of DNA. Enzymes such as helicase and topoisomerase are involved in unwinding the double-stranded DNA
  • Step is important in allowing RNA to access the DNA
• Template Strand (antisense strand) is one of the two nucleotide DNA strands. A single antiparallel and complementary RNA strand is produced from the template strand.
• RNA is synthesized by DNA-dependent RNA polymerase
  • Located genes by searching for promoter regions: specialized DNA regions for recognition
  • RNA-Polymerase II: main enzyme for eukaryotes in transcribing mRNA
    • Binding site is known as the TATA box
  • Transcription factors: help the RNA polymerase locate and bind to promoter region of DNA which helps transcription start.
  • Does not require a primer to start
• Three types of RNA polymerases in eukaryotes:
  • RNA polymerase I: located in the nucleolus and synthesizes rRNA
  • RNA polymerase II: in nucleus and synthesizes hnRNA (pre-processed mRNA) and snRNA (small nuclear RNA)
  • RNA polymerase III: in nucleus and synthesizes tRNA and some rRNA
• RNA polymerase travels along template strand in the 3’ to 5’ direction, this allows for the construction of transcribed mRNA in the 5’ to 3’ direction.
  • Does not proofread work
  • Coding Strand of DNA is the one that is not used as a template for mRNA. It has the same codons as the mRNA strand (Except the t’s are replaced by u’s)
• Numbering system is used to identify the location of important bases in a DNA strand
  • First base transcribed from DNA to RNA is defined as “+1”
  • Bases to the left are negative and to the right are positive.
  • TATA box is usually around -25
• Transcription continues along coding region until a termination sequence or stop signal is reached
  • DNA double helix reforms
  • Heterogenous nuclear RNA (hnRNA) is the primary product of transcription. mRNA is derived from this

Posttranscriptional Processing

• Before hnRNA can leave nucleus and be translated to a protein it must undergo three processes to allow it to interact with the ribosome and survive the conditions of the cytoplasm.

Splicing: Introns and Exons

• Splicing is maturation process that removes non-coding sequences (introns) and then ligate coding sequences (exons) together.
• Accomplished by the spliceosome
  • Small nuclear RNA (snRNA) molecules couple with small nuclear ribonucleoproteins (snRNPs).
  • snRNA/snRNP complex recognizes both the 5’ and 3’ splices of the introns
• Noncoding sequences are excised in the form of a lariat (lasso-shaped structure) and then degraded
• Intron functionality is not well known. Hypothesized that they play a role in the regulation of cellular gene expression levels and maintaining the size of the genome.
  • Also hypothesized to allow for rapid protein evolution

5’ Cap

• A 7-methylguanylate triphosphate cap is added on the 5’ end of the hnRNA molecule
• Added during transcription and is recognized as the binding site by the ribosome
• Protects mRNA from degradation in the cytoplasm

3’ Poly-A Tail

• A polyadenosyl (poly-A) tail is added to the 3’ end of the mRNA transcript
• Protects the message from rapid degradation
• Composed of adenine bases
• Think of it as a “fuse” As soon as the mRNA leaves the nucleus, it will start to get degraded from its 3’ end
  • Longer the tail, the more time the mRNA will be able to survive.
• Tail also assist with the export of mature mRNA cells from the nucleus.

Alternative Splicing

• may occur when the primary hnRNA transcript is spliced together in different ways to produce multiple variants of proteins encoded by the same original gene.

1. Gives ability to make many more different proteins from a limited number of genes
2. Also known to function in the regulation of gene expression

Translation

• The mRNA transcript can exit through the nucleus by nuclear pores
• Once in the cytoplasm, mRNA finds a ribosome to begin the process of translation
  • Converting the mRNA transcript into a functional protein

The Ribosome

• Ribosome is composed of proteins and rRNA
  • Has large and small subunits, and these subunits only bind together during protein synthesis.
• Structure of ribosome dictates its main function: bring mRNA message together with the charged aminoacyl-tRNA complex to generate a protein.
• Three binding sites in the ribosome for the tRNA
  • A site (aminoacyl)
  • P site (peptidyl)
  • E site (exit)
• Eukaryotic ribosomes contain four strands of rRNA: 28S, 18S, 5.8S, and 5S
  • Genes for 28S, 18S and 5.8S are found in the nucleolus
  • RNA polymerase I transcribes the 28S, 18S, and 5.8S rRNA’s as a single unit in the nucleolus which results in the formation of a 45S ribosomal precursor RNA
  • 45S pre-rRNA is processed to become the 18S rRNA of the small (40S) ribosomal unit, and the 28S+5.8S rRNAs of the large (60S) ribosomal subunit
  • RNA polymerase II transcribes the 5S rRNA (found in the 60S subunit)
  • 60S and 40S ribosomal subunits join during protein synthesis to form the whole 80S ribosome.
• Prokaryotes have 50S and 30S large and small subunits
  • These assemble to create the complete 70S ribosome
  • Numbers are not additive since it depends on both size and shape

Mechanism of Translation

• Translation occurs in the cytoplasm of cells
• In prokaryotes, the ribosomes start translating before the mRNA is complete
• In eukaryotes, transcription and translation occur at separate times and in separate locations
• Translation occurs in 3 steps: initiation, elongation, and termination

Initiation

• Small ribosomal subunit binds to the mRNA
  • In prokaryotes, the small subunit binds to the Shine-Dalgarno sequence in the 5’ untranslated region of the mRNA
  • In eukaryotes, small subunit binds to the 5’ cap structure
• Charged interior tRNA bind to the AUG start codon within the P site of the ribosome
  • Initial amino acid in prokaryotes: fMet; in eukaryotes: methionine
• Large subunit then binds to the small subunit which forms the initiation complex
  • Assisted by initiation factors (IF) – not always associated with ribosome

Elongation

• 3-step cycle that is repeated for each amino acid added to the protein after methionine
• Ribosome moves in the 5’ to 3’ direction along the mRNA
  • Synthesizes the protein from its amino (N-) to carboxyl (C-) terminus
• Ribosome contains three important binding sites:
  • A site: holds the incoming aminoacyl-tRNA complex
    • Holds the next amino acid that is being added to the growing chain
      • Determined by mRNA codon at A site
  • P site: holds the tRNA that carries the growing polypeptide chain
    • Methionine binds here to start the chain
    • Polypeptide bond is formed when polypeptide is passed from the tRNA in the P site to the tRNA in the A site.
      • Requires peptidyl transferase (large subunit enzyme)
      • GTP is used for energy in the formation of the bond
  • E-Site: where the inactivated (uncharged) tRNA pauses before exiting the ribosome.
    • tRNA quickly unbinds from the mRNA and is ready to be recharged
• Elongation Factors (EF): locate and recruit aminoacyl tRNA along with GTP. Also removes GDP once it is used.
• Some eukaryotic proteins contain signal sequences that tell a specific destination for the protein
  • E.g. – for hormones/digestive enzymes, signal sequence directs ribosome to move to the endoplasmic reticulum.
    • Protein can then be translated directly into the lumen of the rough ER
    • Proteins can then be sent to the Golgi A and be secreted from a vesicle
  • Other signals can direct the proteins to the nucleus, lysosomes or cell membrane

Termination

• Occurs when and of the three stop codons moves into the A site
• Protein called the release factor (RF) binds to the termination codon
  • Causes a water molecule to be added to the polypeptide chain
• Addition of water allows peptidyl transferase and termination factors to hydrolyze the completed polypeptide chain from the final tRNA
• Polypeptide chain is released from the tRNA in the P site and the two subunits dissociate

Posttranslational Processing

• Newly formed (nascent) polypeptide chain needs to be modified to become a functioning protein
• Proper folding is essential step for the final synthesis of a protein
  • Chaperones: proteins whose main function is to assist in the protein-folding process.
• Some proteins are modified by cleavage events: removing sequences of larger inactive proteins to make them active
  • E.g. – Insulin
  • If signal sequences are used, the sequence must be cleaved if the protein is to accomplish its function.
• For proteins with quaternary structure: subunits come together to form function protein
  • E.g. – hemoglobin: 2 alpha and 2 beta chains
• Peptide may have other biomolecules added through:
  • Phosphorylation: addition of a phosphate group by protein kinases
    • Activated or deactivates proteins
    • Most commonly seen with serine, threonine and tyrosine (amino acids)
  • Carboxylation: addition of carboxylic acid groups (serve as Ca-binding sites)
  • Glycosylation: addition of oligosaccharides as the protein passes through ER and Golgi apparatus
    • Helps determine cellular destination
  • Prenylation: addition of lipid groups to certain membrane-bound enzymes

Control of Gene Expression in Prokaryotes

Operon Structure

• Operon: cluster of genes transcribed as a single mRNA
• Jacob-Monod Model: describes the structure and function of operons
  • Operons contain structural genes, an operator site, a promoter site, and a regulator gene
  • Structural Gene: codes for the protein of interest
  • Operator Site: binds a repressor protein
    • Region of DNA that is non-transcribable
  • Promoter Site: place for RNA polymerase to bind
  • Regulator Gene: codes for a repressor protein
• Two types of operons: inducible systems and repressible systems

Inducible Systems

• Repressor is bonded tightly to the operator system
  • Acts a roadblock due to tightness of bond
  • RNA polymerase is unable to get from the promoter to the structural gene
• Negative Control: binding of protein reduces transcriptional activity
• Block can be removed by having an inducer bind to the receptor protein
  • As the concentration of inducer increases, it will pull more copies of the repressor off of the operator region
• This system allows gene products to be produced only when they are needed (pro)
• E.g. – lac operon: contains the gene for lactase, lactose digestion is harder than glucose, so bacteria only want to digest lactose when the lactose concentration is high and the glucose concentration is low.
  • Lac operon is induced by the presence of lactose
• Catabolite activator protein (CAP): transcriptional advisor used by coli when glucose levels are low. This signals that alternative carbon sources should be used
  • Falling levels of glucose cause an increase in the signaling of cAMP, and this cAMP binds to CAP
  • Conformational change is induced in CAP
    • Allows it to bind to the promoter region of the operon, which increases transcription of the lactase gene
  • Known as positive control mechanisms: binding of a molecule increases transcription of a gene

Repressible System

• Allow constant production of a protein product
• Repressor made by regulator gene is inactive until it binds to a corepressor
  • This complex then binds to an operator site to prevent further transcription
• Tend to serve as negative feedback
  • i.e – final structural protein can serve as co-repressor. When its levels get too high, the transcription of it can be stopped.
• E.g- trp operon – tryptophan is the final protein and acts as corepressor when concentrations get too high

Control of Gene Expression in Eukaryotes

• Much more complex than in prokaryotes
• Helps in maintaining the overall functionality of the cell

Transcription Factors

• Transcription-activating proteins that search the DNA and look for specific DNA-binding motifs.
• Have two domains:
  • DNA-Binding Domain: binds to a specific nucleotide sequence in the promoter region or to a response element (sequence of DNA that binds only to specific transcription factors)
  • Activation Domain: allows for the binding of several transcription factors and other regulatory proteins

Gene Amplification

• A transcription complex is capable of maintain a basal transcription level which results in a steady and adequate level of protein encoded
• When the expression of a protein needs to be amplified, Eukaryotic cells accomplish this through enhancers and gene duplication

Enhancers

• Response elements can be found outside the normal promoter region. These elements can be recognized by specific transcription factors in order to enhance transcription levels.
• Enhancer: several response elements grouped together
  • This allows for the control of one gene’s expression by multiple signals
• E.g – Signal molecules (cAMP, cortisol and estrogen) bind to specific receptors. Receptors are transcription factors that bind to their respective response elements within the enhancer
• Enhancer regions in the DNA can be up to 1000 base pairs away from the gene they regulate, or can be within an intron of the gene.
  • Differ from promoter regions since P regions must be within 20 bases of the start of the gene.
  • Enhancer regions increase the likelihood of amplification

Gene Duplication

• Can increase the expression of gene product by duplicating the relevant gene
• Genes can be duplicated in series on the same chromosome, which would yield many copies, in a row, of the same genetic information.
• Genes can be duplicated in parallel by opening the gene with helicase and permitting DNA replication of only that gene.
  • Cells can then continue replicating the gene until hundreds of copies of the gene exist in parallel on the same chromosome.

Regulation of Chromatin Structure

• DNA is packed in the nucleus as chromatin.
• Heterochromatin: tightly packed DNA that appears dark under a microscope.
  • Tight coils make it inaccessible to the transcription machinery (inactive genes)
• Euchromatin: looser and appears light under the microscope (active genes)

Histone Acetylation

• Transcription factors that bind to the DNA can recruit other coactivators such as histone acetylases
• Histone acetylases proteins are involved in chromatin remodeling
  • Acetylation of histone proteins decreases the positive charge of lysine residues and weakens the interaction of the histone with the DNA
    • Results in an open chromatin conformation
• Specific patterns can lead to an increase or decrease in gene expression levels.
• Histone deacetylases: proteins that function to remove acetyl groups from histone
  • Results in a closed chromatin conformation and overall decrease in gene expression

DNA Methylation

• DNA methylases: add methyl groups to cytosine and adenine nucleotides
• Methylation of genes is linked with the silencing of gene expression
• Heterochromatin regions of the DNA are much more heavily methylated