Transcription and Translation

• These lecture notes cover transcription, translation, and basic differences between prokaryotes and eukaryotes.
• The notes help answer Topic 5B Application Activity questions and the Review Quiz.

Learning Objectives

• Explain semi-conservative replication of DNA.
• Describe the roles of DNA polymerase, DNA helicase, and the origin of replication in DNA replication.
• Describe the source of energy for phosphodiester bond formation.
• Predict the complementary DNA sequence given one strand.
• Describe the Central Dogma, explaining the roles of transcription and translation.
• Describe the difference between transcription and DNA replication.
• Define "gene" and use it to describe DNA organization.
• Describe transcription, including the function of RNA polymerase.
• Identify the energy source for transcription.
• Describe the function of the promoter and terminator, and explain how they define a gene's boundaries.
• Transcribe a DNA sequence into mRNA.
• Describe the differences between pre-mRNA and mature mRNA in eukaryotes.
• Identify and describe the three modifications made to pre-mRNA in eukaryotes, and outline the functions of each modification.
• Distinguish between introns and exons.
• Explain why the reading frame is important for translation.
• Use the genetic code table to predict a peptide sequence from an mRNA sequence.
• Identify codons for specific amino acids and for starting and ending translation.
• Identify all molecules needed for translation.
• Describe the structure of tRNA and ribosomes and their roles in translation.
• Describe the process of translation that attaches amino acids in the correct sequence to produce a polypeptide.
• Describe differences in gene expression between prokaryotes and eukaryotes and explain why they exist.

DNA Replication

• DNA replication is the DNA-dependent synthesis of new DNA, occurring every time a cell divides and producing two identical DNA copies.
• The basic DNA replication process involves breaking hydrogen bonds between the parent DNA bases.
• Each parent strand serves as a template for synthesizing a new, complementary DNA strand.
• DNA replication being semi-conservative means each daughter helix has one parental DNA strand and one newly synthesized strand.
• Replication starts at the origin of replication and involves DNA polymerase and helicase enzymes.
• dNTPs (deoxyribonucleotide triphosphates) are the monomers used in DNA replication.

Origin of Replication

• The origin of replication (ori) is a specific DNA sequence where replication starts.
• At the ori, the DNA helix separates as hydrogen bonds break, creating two single-stranded templates.

DNA Polymerase

• DNA polymerase catalyzes the synthesis of a new DNA strand by adding nucleotides to the 3' end of the growing molecule.
• Synthesis proceeds in the 5' to 3' direction.
• Complementary base pairing guides nucleotide selection.
• The enzyme facilitates phosphodiester bond formation between the 3'-OH and the 5'-phosphate through a dehydration reaction.
• Energy for this synthesis comes from hydrolyzing the triphosphate of the incoming nucleotide.

DNA Helicase

• DNA helicase unwinds the DNA double helix.
• DNA helicase unwinding exposes more single-stranded template.

Central Dogma

• The central dogma describes information flow in a cell: how DNA information is decoded.
• Step 1 (Transcription): RNA is synthesized using DNA as the template.
• Transcription is a nucleic acid (nucleotides) to nucleic acid (nucleotides) process.
• Step 2 (Translation): A polypeptide is synthesized using mRNA-encoded information.
• Translation is a nucleic acid (nucleotides) to polypeptide (amino acids) process.
• Transcription occurs one gene at a time, unlike DNA replication.
• A gene comprises a hereditary information unit with a specific nucleotide sequence in DNA.
• Genes possess unique sequences and can be transcribed to produce RNA.
• Many, but not all, genes encode proteins.
• Gene expression refers to how DNA directs protein synthesis.

Transcription

• Transcription defined as DNA-dependent RNA synthesis.
• Like DNA replication, transcription separates two parental DNA strands by breaking H-bonds.
• A single-stranded DNA acts as the template to create a complementary RNA strand.
• Transcription uses complementary base-pairing rules.
• The key enzyme is RNA polymerase (RNAP), and the monomers are NTPs (Ribonucleotide triphosphates).
• Transcription begins at the promoter and ends at the terminator.
• RNA polymerase (RNAP) incorporates new RNA nucleotides onto the 3’ end of a growing RNA molecule.
• RNA polymerase forms phosphodiester bonds
• RNA polymerase requires a single-stranded DNA template.
• RNA polymerase follows base-pairing rules.
• Energy for transcription comes from hydrolyzing the triphosphate group of the incoming nucleotide.
• A promoter is a specific DNA sequence where RNAP (RNA polymerase) binds and begins transcription.
• The promoter tells RNAP where to start.
• A terminator is a specific DNA sequence where RNA polymerase falls off the template and ends transcription.
• The terminator releases the RNA transcript.
• A gene can be defined as the sequence starting at a promoter and ending at a terminator.
• RNA polymerase transcribes a gene to produce a single RNA molecule.
• Only one DNA strand acts as the template.
• During transcription, the template strand is read by RNA polymerase, and RNA is synthesized through complementary base pairing.
• The non-template strand (coding strand) is not read by RNA polymerase. The non-template strand has the same sequence as the RNA produced.
• Transcription Summary: RNA polymerase binds to the promoter, uses the template strand to synthesize RNA, and dissociates from the template at the terminator.
• All RNAs are produced by transcription.
  • mRNA (messenger RNA) carries information for translation. mRNA is translated into proteins.
  • rRNA (ribosomal RNA) is a structural and enzymatic component of the ribosome.
  • tRNA (transfer RNA) guides amino acids to the ribosome during translation.

mRNA Processing in Eukaryotes

• Eukaryotes modify their RNA prior to translation.
• Eukaryotic modification is not done in prokaryotes. Changes are made to ends and interior sequences.
• pre-mRNA is the initial RNA transcript before modifications.
• Mature mRNA is the modified RNA transcript.

Alterations to mRNA ends

• 5’ Cap: A modified guanine (G) nucleotide added to the 5’ end
• The 5' cap protects mRNA from degradation and aids in attachment to the ribosome.
• 3’ Poly-A tail: 50-250 adenine (A) nucleotides added to the 3’ end
• The 3' poly-A tail protects mRNA from degradation and aids in export from the nucleus to the cytoplasm.

mRNA splicing

• Introns are non-coding or intervening sequences (don’t encode protein).
• Exons are coding or expressed sequences (encode protein).
• The spliceosome enzyme cuts out introns from pre-mRNA and joins exons together.
• Splicing produces mature mRNA with a continuous protein-coding sequence.

Genetic Code

• Translation is the synthesis of polypeptide using an mRNA sequence as a template.
• 4 nucleotides encode 20 amino acids.
• 1 letter code could encode 4 AA
• 2 letter code could encode 16 AA
• 3 letter code could encode 64 AA
• A codon is a three-nucleotide sequence of RNA that specifies a particular amino acid.
• Codons are read 5’ to 3’.
• One codon = 3 nucleotides = 1 amino acid

Reading Frame

• The reading frame is the way a cell’s translation machinery groups the mRNA nucleotides into codons.
• Codons do not overlap.
• There are 3 possible frames per sequence, but only one makes sense and encodes the protein.
• 61 of 64 codons encode amino acids.
  • Each codon encodes only one amino acid.
  • More than 1 codon for each amino acid.
  • Synonym codons differ in the 3rd position.
• AUG is the start codon.
  • AUG tells the ribosome where to start translation.
  • AUG defines reading frame groupings.
  • AUG encodes a methionine (met) amino acid.
  • All proteins start with Met.
• Three codons are stop codons.
  • Stop codons do not encode amino acids.
  • Stop codons tell the ribosome where to stop translation.

Structure of a mature mRNA:

• 5’ untranslated region (UTR): Before start codon; not translated, but involved in ribosome binding
• Open reading frame (ORF): Encodes the protein and has no stop codons present
• 3’ untranslated region (UTR): After the stop codon, not translated
• The genetic code is universal
  • Genes can be moved between organisms which highlights the universal nature of the genetic code.

Translation

• Translation involves the RNA-dependent synthesis of a polypeptide.
• The enzyme involved in translation is the ribosome (rRNA).
• The key component in translation is tRNA.
• mRNA acts as the template for translation.
• Amino acids serve as the monomers for translation.
• Translation begins at the start codon and ends at the stop codon.
• Amino acids are linked by peptide bonds.
  • Peptide bonds are created by a dehydration reaction that releases water.
  • Peptide bonds attach the amino group to the carboxyl group.

Ingredients for Translation:

• mRNA carries information (order of amino acids).
• tRNA delivers the amino acids.
• The ribosome (contains rRNA) uses the information in mRNA to attach amino acids in the correct order.
• Amino acids are attached in the correct order through the energy from GTP (like ATP, but with a G base instead of an A).

tRNA

• tRNA delivers amino acid to the ribosome during translation

Structure:

• It is a small RNA molecule.
• Its is due to internal base-pairing.
• 2 key parts
  • Anticodon: A 3 nucleotide sequence that base pairs to a complementary codon in mRNA
  • Amino acid (AA) attachment site: location where amino acid is covalently attached to the tRNA
  • Each tRNA carries only one AA.Which AA depends on anticodon.

Ribosome

• A ribosome is an enzyme that catalyzes peptide bond formation between amino acids.
• Ribosomes contain two subunits (large and small).
• Both subunits are complexes of ribosomal RNA (rRNA) and ribosomal proteins.
• Ribosomes contain several binding sites:
  • One mRNA binding site.
  • Two tRNA binding sites: The A site is where a new tRNA (carrying amino acid) binds. The P site is where tRNA holding a growing polypeptide binds.
• The E (exit) site allows empty tRNA to leave the ribosome.

Functions

• The ribosome holds mRNA and tRNA together.
• The ribosome positions a new amino acid for addition to a growing polypeptide chain.
• The ribosome catalyzes the formation of peptide bonds.

Mechanism of Translation:

• Start codon initiates translation.
• Amino acids are added to growing polypeptide: tRNA delivers a new amino acid to the ribosome by base pairing to the mRNA codon in the A site
• The ribosome (rRNA) creates a peptide bond between a new amino acid and the existing chain.
• The ribosome moves 5’ → 3’ down mRNA to read the next codon.
• The empty tRNA is released.
• The recognition of a stop codon terminates translation.

Prokaryotes vs. Eukaryotes

Similarities:

• Transcription: synthesis of RNA using DNA as a template
• Translation: synthesis of polypeptide using mRNA sequence as a template
• The ribosome is the enzyme for translation.
• Both use the same genetic code.

Differences:

• Same function, different size
• Eukaryotic vs. Prokaryotic ribosomes have different types of rRNA and proteins
• Bacterial ribosomes are susceptible to antibiotics; eukaryotic ribosomes aren’t.
• Transcription and translation are coupled in prokaryotes
  • Translation starts before transcription finishes
  • Allows polyribosomes to form
  • Transcription and translation both occur in the cytoplasm.
  • Transcripts are not processed.
• Transcription and translation are separated in timing and location in eukaryotes
  • Transcription occurs in the nucleus
  • Translation occurs in the cytoplasm
  • Transcripts are processed before translation
  • mRNA must be exported from nucleus.