Chapter 10 - The Expression of Genetic Information: Transcription and Translation

Overview of Gene Expression

• Gene expression involves transcription and translation at the molecular level.

• Transcription produces an RNA copy of a gene (DNA à RNA).

• Translation interprets the nucleotide sequence in mRNA to build a polypeptide with a specific amino acid sequence (RNA à protein).

• In eukaryotes, an additional step of RNA modification is used.

• The central dogma describes information flow from DNA to RNA to protein.

• Some genes encode functional RNA molecules instead of polypeptides.

• Retroviruses use an RNA template to make DNA (RNA à DNA), which is an exception to the central dogma.

• Genes provide a "blueprint" for an organism's characteristics.

• Most genes are structural genes coding for polypeptides.

• One or several polypeptides function as a protein, playing a role in the cell.

• Protein activities determine cell structure and function, ultimately determining organism traits.

Transcription

• Transcription copies a discrete unit of information from DNA into RNA.

• DNA is not altered, allowing repeated use as an information source.

• A gene is an organized unit of nucleotide sequences transcribed into RNA, forming a functional product (protein or RNA).

• Important sequences include the promoter, transcribed region, terminator, and regulatory sequences.

• Transcription occurs in 3 stages: initiation, elongation, and termination.

  • Initiation: Promoter functions as a recognition site.

    • In bacteria, sigma factor binds to RNA polymerase, facilitating binding to the promoter.

    • DNA strands are separated to form an open complex.

  • Elongation: RNA polymerase synthesizes RNA.

    • The template strand is used as a template, and the opposite strand is the coding strand.

    • The transcript is synthesized in the 5’ to 3’ direction.

• 5’ —> 3’

  • termination: RNA polymerase reaches a terminator, releases the transcript, and dissociates from DNA

• Eukaryotes and bacteria share basic transcription features (promoters, initiation, elongation, termination).

• Eukaryotic transcription involves more complex protein components.

• Eukaryotes have 3 forms of RNA polymerase (I, II, III), while bacteria have one.

  • RNA polymerase II transcribes mRNA from protein-coding genes.

  • RNA polymerases I and III transcribe non-coding genes (tRNAs and rRNAs).

RNA Modifications in Eukaryotes

• In eukaryotes, pre-mRNA (immature precursor) is processed into mature mRNA.

• Pre-mRNAs undergo 3 key modifications:

  • 5’ cap addition: A modified guanine is covalently attached to the 5’ end.

    • The 5’ cap is recognized by proteins, needed for mRNA to exit the nucleus and bind the ribosome.

  • 3’ poly A tail addition: Added to the 3’ end after transcription (via enzyme activity).

    • The poly A tail increases mRNA stability in the cytosol.

  • Splicing: Introns (intervening regions) are removed, and exons (expressed regions) are connected.

• Introns are found in many eukaryotic genes.

• An average human gene has about 9 introns (ranging from dozens to over 100,000 nucleotides).

• The spliceosome removes introns precisely.

• The spliceosome is composed of snRNPs (small nuclear RNA + proteins).

• Alternative splicing allows complex eukaryotes to use the same gene to make different proteins.

• Splicing steps 3 and 4 are catalyzed by an RNA component (ribozyme activity).

Translation and the Genetic Code

• The genetic code specifies the relationship between mRNA nucleotide sequence and polypeptide amino acid sequence.

• The code is read in groups of 3 nucleotides called codons.

• There are 64 different codons:

  • 1 start codon (AUG)

  • 3 stop codons (UAA, UAG, UGA)

  • 61 codons specify amino acids

• The code is redundant; more than one codon can specify the same amino acid.

• Key components for translation include the ribosomal-binding site, start codon, coding sequence, and stop codon.

• The start codon defines the reading frame (groups of 3 nucleotides read as codons).

• Codons are read sequentially and non-overlapping in the 5’ to 3’ direction.

5' \rightarrow 3'

• tRNAs translate the mRNA nucleotide sequence into the polypeptide amino acid sequence.

• tRNAs contain an anticodon (3-base sequence) complementary to an mRNA codon.

• Different tRNAs have different anticodon sequences, each carrying a specific amino acid.

• mRNA is read in the 5’ to 3’ direction, and the polypeptide is synthesized from the N-terminus to the C-terminus.

The Machinery of Translation

• Many components are necessary for translation, requiring substantial cellular energy.

• tRNAs share common features:

  • 2-D cloverleaf structure with 3 loops and a stem.

  • Anticodon located in the middle loop.

  • 3’ single-stranded region is the amino acid attachment site.

  • 3-D structure involves additional folding.

• Cells make many different tRNAs, each encoded by a different gene.

• tRNAs are named according to the amino acid they carry (e.g., tRNASer carries serine).

• Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to tRNA molecules.

• Cells make 20 distinct types of aminoacyl-tRNA synthetases, each recognizing one of the 20 different amino acids.

• Each enzyme is named for the specific amino acid it attaches (e.g., alanyl-tRNA synthetase recognizes alanine).

• Aminoacyl-tRNA synthetases catalyze reactions involving an amino acid, a tRNA molecule, and ATP.

• A tRNA with its amino acid attached is called a charged tRNA.

• Ribosomes are the sites of translation.

• Prokaryotic and eukaryotic ribosomes differ but share common structural features.

• Both are large complexes formed from a small and a large subunit.

• Subunits contain one or more rRNA molecules and numerous proteins.

• Ribosomes contain 3 discrete sites where tRNA may be located: the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site.

• Components for translation arose in the ancestor of all living species.

• The gene for the small subunit rRNA is found in all species.

• Generally, if two species diverged a long time ago, their gene sequences are quite different; if they diverged recently, their gene sequences are more similar.

The Stages of Translation

• Translation occurs in 3 stages: initiation, elongation, and termination.

  • Initiation: mRNA, the first tRNA, and the ribosomal subunits assemble into a complex.

  • Elongation: The ribosome moves in the 5’ to 3’ direction from the start codon towards the stop codon, synthesizing a polypeptide.

• 5’ —> 3’

  • Termination: the ribosome reaches a stop codon and the the complex disassembles, releasing the polypeptide

• Initiation factors are proteins that help assemble the mRNA, tRNA, and ribosome into a functional complex; GTP hydrolysis provides energy.

• In bacteria, the first tRNA carries a modified methionine and resides in the P site.

• Initiation of translation in eukaryotes differs:

  • Instead of a ribosomal-binding sequence, mRNAs have the 5’ guanosine cap, which is recognized by proteins that promote mRNA binding to the small subunit.

  • The position of the start codon is more variable.

  • The initiator tRNA carries a regular methionine (not a modified formyl-methionine).

• The elongation stage involves the covalent bonding of amino acids to each other via codon/anticodon recognition.

• Elongation factors hydrolyze GTP to provide energy to bind tRNA to the A site.

• Termination occurs when a stop codon is reached.

• The 3 stop codons (UAA, UAG, UGA) are recognized by a release factor protein, not a tRNA.