Molecular Genetics Basics

Chapter 17: Transcription, RNA Processing, and Translation

• This chapter examines how genetic information in genes directs the synthesis of RNA and proteins.
• It explores how DNA is transcribed into RNA, how eukaryotes process RNA, and how messenger RNA is translated into proteins.
• It also looks at the structure and function of transfer RNA and ribosomes.

Learning Objectives

• Describe the steps of transcription.
• Describe how primary transcripts are processed.
• Describe the roles of ribosomes, mRNA, and tRNAs in translation.
• Analyze the structure and function of transfer RNA.
• Explain the events of translation initiation, elongation, termination, and post-translational modification.

Part I: Transcription

• Transcription utilizes RNA polymerase to synthesize RNA from a DNA template.
• RNA polymerase moves along the DNA template strand in the 3' to 5' direction, synthesizing the RNA transcript in the 5' to 3' direction.
• The RNA transcript is complementary to the template and identical to the nontemplate (coding) DNA strand, with uracil (U) replacing thymine (T).

Review: Differences between DNA and RNA

• DNA is a double helix while RNA is a single stranded molecule.
• DNA contains the sugar deoxyribose and bases Adenine, Guanine, Cytosine and Thymine.
• RNA contains the sugar ribose and bases Adenine, Guanine, Cytosine and Uracil.

Review: What are DNA's functions?

• DNA stores genetic information in the form of genes, which can come in different versions.
• DNA regulates when and where genes are expressed.
• DNA transmits genetic information to the next generation through replication.
• Replication must be exact, but errors may occur, leading to mutations which can have varying impacts.

Transcription versus Translation

• Transcription converts DNA information into RNA.
• Translation converts RNA information into proteins.

Functions of DNA and RNA

• DNA is genetic material passed from parents to offspring and contains instructions for making proteins.
• RNA is essential for protein synthesis and plays a role in gene expression regulation.

Central Dogma of Molecular Biology

• Information flows from DNA to RNA to polypeptide (protein).
• Transcription converts DNA to RNA.
• Translation converts RNA to protein.
• Ribosomes are the site of translation.

The Genetic Code

• A sequence of three nucleotides (codon) codes for a specific amino acid.
• Multiple codons can code for the same amino acid.
• The genetic code is read in groups of three nucleotides (codons) in the correct reading frame.
• The genetic code is universal, meaning it is the same for all life forms.

What do we need for Transcription?

• DNA template strand, RNA polymerase, transcription factors.

RNA Polymerase

• Separates DNA strands and synthesizes mRNA in the 5′ to 3′ direction.
• RNA polymerase travels along the DNA, reads the nontemplate strand and builds the complementary mRNA strand.

Eukaryotic Transcription Initiation

• Eukaryotic cells utilize TATA boxes within promoters for transcription factor recognition and subsequent RNA polymerase binding.

Elongation

• RNA polymerase continues adding bases to the growing mRNA strand in a complementary fashion to the template strand.

Termination

• RNA polymerase transcribes a terminator sequence in the DNA, then mRNA and polymerase detach.
• Prokaryotic cells = mRNA is ready for use after transcription.
• Eukaryotic cells require further processing of mRNA called pre-mRNA which occurs after transcription.

mRNA processing in eukaryotes

• Pre-mRNA has introns (noncoding regions) and exons (coding regions).
• Introns are removed and exons are joined together via splicing to form mRNA.
• A 5' cap and 3' poly-A tail are added to the mRNA transcript.
• Modification enhances mRNA stability, the export of mRNA from the nucleus, and proper binding to ribosomes.
  • 5' cap = modified guanine.
  • 3' poly-A tail = 50-520 adenine molecules.

RNA Splicing

• Introns are removed and exons are spliced together to form the mature transcript in a process called splicing.
• This results in a smaller, more compact, mature mRNA molecule ready to be transported from the nucleus.

Translation

• mRNA, tRNA, and ribosomes are required for protein synthesis.
• Ribosome = site of translation.
• tRNA carries amino acids to the ribosomes.
• mRNA codes for amino acids which tRNA translates to proteins.
• mRNA carries a sequence of codons that code for amino acids for forming polypeptide chains.
• tRNA contains an anticodon which is complementary to the mRNA codon and carries a specific amino acid.

tRNA Role

• Specific to each amino acid.
• Transfers amino acids to ribosomes.
• Contains an anticodon that pairs with the complementary mRNA codon.

Ribosome Components

• Ribosomes have an EPA site for tRNA binding, where codon-anticodon interactions occur to bring the correct amino acid into the growing polypeptide chain.

Translation Steps

• Initiation, elongation, termination.

Initiation

• The small ribosomal subunit binds to the mRNA's recognition sequence.
• A Met (methionine) tRNA binds to the AUG start codon.
• The large ribosomal subunit completes the initiation complex.

Elongation

• An incoming tRNA carries an amino acid and binds to the next codon in the A site.
• A peptide bond forms between the amino acids in the P and A sites.
• The ribosome moves one codon along the mRNA, moving the tRNA to the E site, where it then disassociates from the ribosome, freeing the tRNA to carry a new amino acid.

Termination

• When the ribosome encounters a stop codon, a release factor enters the A site.
• The polypeptide chain is released. The mRNA and ribosomal subunits separate.

Signals that start Initiation and Termination in Transcription and Translation

• Transcription signals:
  • Initiation: promoter
  • Termination: terminator.
• Translation signals:
  • Initiation: start codon
  • Termination: stop codon.

Compare gene expression in prokaryotes vs. eukaryotes

• Prokaryotes: transcription and translation occur in the cytoplasm. Genes lack introns, and no mRNA modification occurs.
• Eukaryotes: transcription occurs in the nucleus and translation in the cytoplasm. Genes contain introns, and mRNA must be processed before leaving the nucleus.

Possible effects of mutations on gene expression

• Mutations are changes in DNA sequence.
• Mutations can cause silent mutations (no change in amino acid sequence), missense mutations (change in amino acid sequence), or nonsense mutations (early stop codon).
• Frame shift mutations (insertion or deletion of a nucleotide) cause alterations in subsequent amino acids.

Missense Mutation causes Sickle-Cell Anemia

• A nucleotide substitution changes the amino acid, from glutamic acid to valine, causing abnormal polypeptide structure, and thus abnormal hemoglobin function in red blood cells.