Molecular Biology: Translation Process Quiz

Nucleic Acids - Structure & Function

• DNA and RNA are long polymers
• Each nucleotide is a monomer
• Each nucleotide consists of:
  • A nitrogeneous heterocyclic base
    • Purine
    • Pyrimidine
  • A 5-carbon sugar
    • Ribose
    • Deoxyribose
  • A phosphoryl group
• Both the base and sugar have ring structures
• Sugars are numbered with 'prime' (')
• Bases are numbered without 'prime'
• A covalent bond between sugar and phosphoryl is a phosphoester bond
• A bond between base and sugar is a β-N-glycosidic linkage joining the 1' carbon of sugar and N of the base
• Nitrogenous bases are heterocyclic amines
• Cyclic compounds with at least one nitrogen atom in the ring
• Purines are double rings: a 6-member ring fused to a 5-member ring
  • Adenine (A)
  • Guanine (G)
• Pyrimidines consist of a single 6-membered ring
  • Cytosine (C)
  • Uracil (U) - found in RNA
  • Thymine (T) - found in DNA
• DNA and RNA are polymers of nucleotides.
• Nitrogen base is β-attached to:
  • Ribose (RNA)
  • Deoxyribose (DNA)
• The sugar is phosphorylated at carbon 5'.

Prokaryotic Chromosomes

• Prokaryotes are single-celled organisms without membrane-bound organelles
• Chromosomes are DNA pieces with genetic instructions (genes)
• Prokaryotic chromosomes are:
  • Single, circular/loop DNA molecules
  • Supercoiled
  • Attached to the inner membrane of the prokaryote
  • Located in a nucleoid region of the cytoplasm (not a membrane-bound nucleus)

Eukaryotic Chromosomes

• Eukaryotic chromosomes are linear and vary in number and size
• They have a true nucleus surrounded by a nuclear membrane
• DNA is wrapped around histone proteins to form nucleosomes
• DNA appears as "beads on a string"
• Nucleosomes coil into 30nm fibres
• Further coiling leads to 300nm fibres

RNA Structure

• RNA's backbone is composed of S-P-S-P
• RNA is primarily single-stranded;
• Ribose replaces deoxyribose
• Uracil replaces thymine
• Base pairing (A=U) and (C=G) forms double-stranded regions (viruses)

DNA Replication

• DNA must be replicated before cell division
• Each daughter cell needs a copy of each gene
• The replication process must produce an accurate copy of the original genetic information
• Mistakes in replication can cause lethal mutations
• Replication is catalyzed by DNA polymerase
• The molecule has an original/parent strand and newly synthesized strand
• Replication is semiconservative, resulting in two new DNA helices each with one original strand
• Bacterial DNA replication begins at a unique sequence (replication origin)
• Replication moves bidirectionally, at a rate of 500 nucleotides/second
• The position where nucleotides are added is the replication fork.
• There are two replication forks that move in opposite directions

Details of DNA Replication

• Helicase separates the strands by breaking hydrogen bonds
• Positive supercoiling is relieved by topoisomerase
• Single-strand binding proteins prevent strands from rejoining (reannealing)
• Primase synthesizes RNA primers needed for DNA polymerase
• DNA polymerase III elongates the new strands
• DNA polymerase I removes RNA primers and replaces them with DNA nucleotides
• DNA ligase joins together Okazaki fragments on the lagging strand

DNA Polymerase Reaction

• DNA polymerase III reads parental DNA/template and creates a complentary strand
• A pyrophosphate group is released during the polymerization
• A new phosphoester bond is formed between the 5' phosphoryl group and the 3'−OH group of the existing nucleotide. This is a 5' to 3' direction.

DNA Replication - Influencing Factors

• The two DNA strands are antiparallel.
• DNA polymerase III can only work in the 5' to 3' direction
• Small RNA primers are required for a starting point of DNA replication

DNA Replication - Leading Strand

• A single RNA primer is produced at the replication origin
• DNA Polymerase III continuously adds nucleotides in the 5' to 3' direction

DNA Replication - Lagging Strand

• Many RNA primers are produced as the replication fork moves along the molecule
• DNA polymerase III catalyzes elongation of the new strand in the 5' to 3' direction
• Okazaki fragments are formed, and then joined by DNA ligase
• The process repeats with another primer made at a new location of the replication fork
• RNA primers are removed, and the gaps are filled by DNA polymerase I
• The fragments are sealed by DNA ligase

Replication Fork - Detailed View

• The lagging strand is synthesized discontinuously and in the opposite direction to the replication fork movement.
• Various enzymes are involved in the process, including DNA polymerase III, DNA polymerase I, primase, ligase, helicase, single-strand binding proteins and topoisomerase.
• Okazaki fragments are formed during synthesis on the lagging strand, which are later joined together by the DNA ligase enzyme.

Central Dogma

• In cells, genetic information contained in DNA flows in one direction to RNA to protein
• Transcription is making a copy of a strand of DNA
• Translation is converting the information from one language of bases to another of amino acids

Classes of RNA Molecules

• Messenger RNA (mRNA): a complementary copy of a gene that directs the amino acid sequence of proteins
• Ribosomal RNA (rRNA): a structural and functional component of ribosomes, forming ribosomes by reacting with proteins. It has 3 types in prokaryotes & 4 types in eukaryotes
• Transfer RNA (tRNA): transfers amino acids to the site of protein synthesis

Transfer RNA (tRNA)

• There is at least one tRNA for each amino acid incorporated into a protein
• tRNA is single-stranded and typically about 80 nucleotides long
• The overall structure is a cloverleaf
• It has intrachain hydrogen bonding between A=U and C=G bases; contains rare bases like D, T, Y, and Ψ
• The 3' end of the molecule has a conserved CCA-3' sequence for amino acid binding

Aminoacyl tRNA Synthetase

• It catalyzes the attachment of a tRNA molecule to its respective amino acid.
• There is at least one aminoacyl tRNA synthetase for each amino acid.

Protein Synthesis

• Protein synthesis is called translation
• It is carried out on ribosomes which are constructed from rRNA and proteins.
• mRNA is translated 5' to 3'
• Protein synthesis occurs in multiple places on a single mRNA molecule, forming a polysome
• tRNA binds specific amino acids by aminoacyl tRNA synthetase enzyme
• tRNA recognizes its complementary codon on the mRNA

Genetic Code

• Degenerate/redundant: more than one codon can code for the same amino acid
• Specific: each codon specifies a particular amino acid
• Nonoverlapping & commaless: codons are read one after another without gaps
• Universal: all organisms use the same genetic code
• All 64 codons have meaning; 61 for amino acids and 3 for "stop" signals
• Multiple codons for an amino acid often share common bases

Ribosomes

• Ribosomes are complexes of rRNA and proteins
• Each ribosome has two subunits: a small subunit and a large subunit;
• Small subunit contains one rRNA and 33 proteins
• Large subunit contains 3 rRNA and 49 proteins
• Many ribosomes on one mRNA comprise a polysome with many copies of the protein made simultaneously

Stages of Transcription

• Initiation: RNA polymerase binds to the promoter region of the DNA
• Elongation: RNA polymerase moves along the template strand, synthesizing a complementary RNA molecule
• Termination: RNA polymerase releases the newly formed RNA molecule

Post-Transcriptional Processing

• Prokaryotes release a mature mRNA at the end of termination
• Eukaryotic mRNA is a primary transcript that needs posttranscriptional modification which include
  • A 5' cap structure is added
  • A 3' poly(A) tail (100-200 adenine nucleotides) is added by poly(A) polymerase
    • Required for efficient translation
    • Protects from degradation

RNA Splicing

• RNA splicing is the removal of portions (introns) from primary mRNA that are not protein coding
• Bacterial prokaryotic chromosomes are continuous; mRNA contains entire DNA sequence
• Eukaryotic chromosomes are discontinuous, forming introns within the gene sequences that do not code for amino acids
• Spliceosomes, composed of snRNPs and other proteins, recognize intron-exon boundaries and stabilize the splicing complex

Mutations, UV Light, and DNA Repair

• Mutations are mistakes introduced into the DNA sequence of an organism
• Mutations can be silent (no change in the protein sequence) or have negative effects on the organism – Mutagens are chemicals causing changes in DNA; also carcinogens
• UV causes covalent linkage of adjacent pyrimidine bases (thymine dimers)
• Xeroderma pigmentosum is a genetic disorder characterized by increased sensitivity to UV radiation which can cause severe skin-burning and cancer

Type of Mutations

• Point mutations involve a single base substitution. These can be silent, missense, or nonsense.
• Frameshift mutations are due to the insertion or deletion of a base—this shifts the reading frame of the codons, changing all subsequent nucleotides.

Recombinant DNA

• Recombinant DNA is synthetic DNA containing segments from more than one source; it joins two different DNA molecules to create a hybrid.
• This technology is made possible by restriction endonucleases (bacterial enzymes that cut DNA at specific nucleotide sequences) and the enzyme ligase.
• Three key elements in forming recombinant DNA are
  • A DNA molecule that will have the new DNA segment.