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Questions and Answers
What is the role of aminoacyl-tRNA synthetases in protein synthesis?
What is the role of aminoacyl-tRNA synthetases in protein synthesis?
Which statement accurately describes the structure of tRNA?
Which statement accurately describes the structure of tRNA?
During the initiation of protein synthesis, what crucial function do ribosomes perform?
During the initiation of protein synthesis, what crucial function do ribosomes perform?
What occurs during the termination phase of protein synthesis?
What occurs during the termination phase of protein synthesis?
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What facilitates ribosome movement during translation?
What facilitates ribosome movement during translation?
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What role does the initiating factor IF2 play during translation initiation in E. coli?
What role does the initiating factor IF2 play during translation initiation in E. coli?
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Which site does the peptidyl-tRNA move to after peptide bond formation during elongation?
Which site does the peptidyl-tRNA move to after peptide bond formation during elongation?
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What is the primary structure of a protein defined by?
What is the primary structure of a protein defined by?
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How does the ribosome read mRNA during translation?
How does the ribosome read mRNA during translation?
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Which of the following accurately describes the role of release factors RF1 and RF2 in E. coli?
Which of the following accurately describes the role of release factors RF1 and RF2 in E. coli?
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What is the role of the Shine-Dalgarno sequence in mRNA during the initiation of translation?
What is the role of the Shine-Dalgarno sequence in mRNA during the initiation of translation?
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What is the function of elongation factors EF-Tu and EF-G in E. coli?
What is the function of elongation factors EF-Tu and EF-G in E. coli?
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What does the presence of multiple ribosomes on a single mRNA molecule indicate?
What does the presence of multiple ribosomes on a single mRNA molecule indicate?
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What is the primary role of the 5’ cap structure added to eukaryotic mRNA?
What is the primary role of the 5’ cap structure added to eukaryotic mRNA?
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Which of the following correctly describes the structure of transfer RNA (tRNA)?
Which of the following correctly describes the structure of transfer RNA (tRNA)?
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What is the function of aminoacyl-tRNA synthetases in protein synthesis?
What is the function of aminoacyl-tRNA synthetases in protein synthesis?
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What signals the termination of protein synthesis during translation?
What signals the termination of protein synthesis during translation?
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What is the primary role of ribosomes during translation?
What is the primary role of ribosomes during translation?
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Which component is crucial for the formation of the 5’ cap on eukaryotic mRNA?
Which component is crucial for the formation of the 5’ cap on eukaryotic mRNA?
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What happens to poly(A) tails as eukaryotic mRNA ages in the cell?
What happens to poly(A) tails as eukaryotic mRNA ages in the cell?
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During splicing, which of the following is formed as a result of the reaction between the 2’-OH of an adenine nucleotide and the 5’ phosphate of a guanine nucleotide?
During splicing, which of the following is formed as a result of the reaction between the 2’-OH of an adenine nucleotide and the 5’ phosphate of a guanine nucleotide?
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What is the significance of alternative splicing in eukaryotic cells?
What is the significance of alternative splicing in eukaryotic cells?
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What initiates the assembly of amino acids during the translation process?
What initiates the assembly of amino acids during the translation process?
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Study Notes
Gene Definition
- Definitions vary depending on the context.
- Geneticists define a gene as a discrete unit of inheritance passed between generations, determining traits (Gregor Mendel).
- Biochemists define a gene as the sequence of bases that defines a functional protein.
- Protein-coding sequences are flanked by regulatory DNA sequences that do not encode amino acids.
- Some genes give rise to non-protein-encoding RNA molecules.
- All cellular RNAs are transcribed from DNA templates.
Gene Number
- Fruit flies have approximately 16,000 genes.
- Human genomes have approximately 20,000-25,000 genes (the number is still being refined as gene prediction becomes more accurate).
- Biological complexity is not directly related to the number of genes.
Central Dogma of Molecular Biology
- DNA → mRNA → Protein
- Central Dogma outlines the flow of information in gene expression.
DNA to Protein Overview
- DNA information is encoded in a sequence of four bases.
- The DNA sequence directs the assembly of 20 amino acids forming a specific protein sequence in a correct order
- The four bases code for 20 amino acids using a three-base code (triplet codon).
- DNA resides within the nucleus and protein synthesis takes place in the cytoplasm.
- Copies of the DNA code are sent to the cytoplasm as messenger RNA (mRNA).
Prokaryotic vs. Eukaryotic Genes
- Prokaryotic genes: arranged closely together.
- Eukaryotic genes: their coding regions are interrupted by non-coding segments (introns).
- Eukaryotic coding regions are exons.
- Eukaryotic genes can have one to several hundred introns, which can range in length (from 50-20,000 nucleotides).
- Exons are usually shorter (~150 base pairs).
Gene Ends
- Promoter region: located at the 5' end of a gene, which is required for transcription.
- Terminator region: located at the 3' end of a gene, which triggers the end of transcription.
- The first template nucleotide is indicated as +1 and the 5' end of this nucleotide is -1.
Prokaryotic Gene and mRNA Structure
- The 5' UTR contains signals for translation initiation.
- The 3' UTR contains signals for translation termination.
Sense and Antisense DNA Strands
- Template strand: referred to as the antisense or noncoding strand.
- Coding strand: has the same sequence as the transcribed RNA.
RNA Polymerase
- mRNA is synthesized by DNA-dependent RNA polymerase.
- Synthesis of mRNA requires the separation of the double-stranded DNA into a single stranded template.
- RNA synthesis proceeds in the 5' to 3' direction, and RNA polymerase can initiate new chains without a primer.
mRNA Synthesis
- Synthesis uses nucleoside triphosphates (e.g., ATP, CTP, GTP, UTP).
- New chain of mRNA is started from the 5' end.
- The incoming nucleotide attaches to the 3' end.
RNA Polymerase (Mechanism)
- DNA polymerase moves along the DNA chain separating the molecule.
- DNA strand unwinds to expose the template strand of the double-helix.
Stages of Gene Transcription in E. coli
- The three stages of gene transcription are: initiation, elongation, and termination.
- Initiation: RNA polymerase binds to the promoter region which is located on the DNA molecule, which defines the gene.
- E coli promoter has two conserved regions: Pribnow box centered at nucleotide -10 and another region at -35
RNA Polymerase (E. coli)
- E. coli RNA polymerase is a protein complex with subunits a₂ββ'ω.
- A sigma subunit (σ) allows holoenzyme binding to consensus sequences and starts the initiation process.
- Specific roles for different sigma factors that identify different promoters are responsible for expression
- The sigma unit, which initiates the transcription process, dissociates from the core enzyme complex (αββ'ω) to allow the core enzyme to carry out the polymerase activity of joining nucleotides to form mRNA
RNA Polymerase (General)
- RNA polymerase is processive, meaning it doesn't detach from the template molecule during the transcription process.
- Transcription is rapid (20-50 nucleotides/second), which is slower than DNA replication (~1,000 nucleotides/second).
- Transcription has a higher error rate (1 in 104) but the rate of error is tolerable because genes are copied several times.
- The genetic code is redundant (multiple codons can specify the same amino acid).
Termination of Transcription
- Two mechanisms of transcription termination exist: 1- Internal base pairing of GC base pairs forms a stem loop structure (stable) preventing further elongation. 2- Rho factor attaching to new mRNA, unwinding the RNA-DNA duplex and releasing the mRNA.
Eukaryotic RNA Polymerase
- Eukaryotes have three different forms of RNA polymerases (I, II, and III).
- Gene expression is highly regulated.
- Primary transcript undergoes post-transcriptional modifications.
- Eukaryotic RNA polymerase has to negotiate the histones that are not totally stripped away from the DNA template; this mechanism is not completely understood.
Major Modifications of Eukaryotic mRNA
- 5' capping
- Addition of a polyA-tail to the 3' end.
- Slicing to remove introns
5' Cap of Eukaryotic mRNAs
- Consists of 7-methylguanosine (m7G).
- Added to the initial (5') nucleotide when the transcript is about 30 nucleotides long in eukaryotes
- Marks the 5’ end and identifies the eukaryotic translation start site
- mRNA needs to have this cap, so it is required.
- Enzymes are involved in removal of leading phosphate group in 5’ mRNA terminal tri-phosphate.
- mRNA capping steps involve the guanylyltransferase enzyme and requires GTP energy.
- A guanosine-7-methyltransferase adds a methyl group to the guanine.
- Further methylations are added to nucleotides one and two.
- Protecting mRNA from degredation.
Poly(A) Tails
- Mature eukaryotic mRNAs have 3'-ends terminating in poly(A) tails of approximately 250 nucleotides.
- Enzymatically added from ATP.
- Not required for mRNA translation.
- The tail shortens as the transcript ages in cells; this suggests a protective role for the tail.
Splicing Removes Introns
- Eukaryotic genes differ from prokaryotic genes in that eukaryotic genes contain interspersed, unexpressed regions within the genome.
- Primary transcript (pre-mRNA) is larger than mature proteins.
- Intervening sequences (introns) are excised.
- The expressed sequences (exons) are joined together.
Steps in mRNA Production
- Steps in the production of mature mRNA:
- Primary transcript production.
- 5' capping and polyadenylation
- Slicing (removal) of introns
- Splicing to join together the exons.
Exons are Sliced
- Exon-intron junctions have high homology to invariant GU at the 5' intron boundary and AG at the 3' intron boundary
- Other signals also control the splicing site
- Splicing involves two transesterification reactions.
Mechanism of Splicing
- 2'-OH group from an adenine nucleotide in the intron chain attacks the 5' phosphate of the G nucleotide in the intron chain causing it to break.
- The 3'-OH (exon 1) attacks the 5' end of the exon 2.
- Joining the exons, without energy requirement.
Splicing Mechanism and Significance
- The splicing reaction is catalyzed by a complex protein-RNA molecular structure called the spliceosome.
- The RNA molecules are called small nuclear RNAs (snRNAs), and when complexed with proteins, they are called small nuclear ribonucleoproteins.
- The spliceosome is a complex machine made up of over 300 polypeptides.
- It is highly dynamic meaning its parts come together and go apart at different stages.
- Splicing allows one gene to produce several different proteins with different functions (alternative splicing); rapid protein evolution occurs.
Protein Synthesis - Translation
- Translation is the process of converting the nucleotide language of mRNA into the amino acid language of protein.
- Transfer RNAs (tRNAs) are used to bring amino acids to mRNA.
- Translation occurs on ribosomes, which are complex organelles (RNA and proteins) found in the cytoplasm.
- Ribosomes move along the mRNA molecule and assemble amino acids into a protein.
The Genetic Code
- A triplet codon corresponds to a particular amino acid.
- The genetic code is nearly universal (with minor exceptions).
- Codons are used in protein synthesis.
Genetic Code (Key points)
- 61 codons specify amino acids (degenerate).
- 3 codons signal the termination of polypeptide synthesis (stop codons).
- AUG (and GUG) are frequently start codons.
Using the Genetic Code
- Read the code (on a chart) by reading from the center (bases) to the outside (amino acids).
Name the Amino Acids
- Given the codons (AAA, AUA, CAU, GGG, and UCA), identify the corresponding amino acids.
The Genetic Code (Additional Points)
- When several codons exist for a single amino acid, these codons show strong relationships and mostly vary in their third base.
- Codons for similar amino acids tend to be similar.
Peptide Synthesis - Basic Chemistry
- Joining two amino acids together requires energy.
- Activation of amino acids links each amino acid to its specific transfer RNA (tRNA).
- Aminoacyl-tRNA synthetases are a family of enzymes that activate amino acids and recognize specific tRNAs.
- This linking happens between the –OH group on the 3' end of the tRNA, and the -COOH of an amino acid group.
Transfer RNA (tRNA)
- tRNAs are small RNA molecules (~less than 100 nucleotides).
- They have a clover-leaf structure stabilized by internal base pairing forming loops.
- The anticodon is located on the tRNA and is complementary to a specific mRNA codon.
- tRNAs have a terminal CCA sequence linked to its amino acid.
Ribosomes
- Ribosomes are small organelles involved in protein synthesis.
- Ribosomes are made up of ribosomal RNA (rRNA).
- Ribosomes contain specific binding sites for tRNA molecules.
- Ribosomes help mediate interactions among other, non-ribosomal proteins, that initiate, elongate, and terminate polypeptide chain.
- Ribosomes catalyze peptide bond formation during protein synthesis.
- Ribosomes move efficiently along the mRNA molecule to translate codons in a sequential method (reading frame).
Ribosomal RNA (rRNA)
- Eukaryotic ribosomes are larger than prokaryotic ribosomes.
- rRNA molecules are highly folded and compacted due to internal base pairing.
Initiation of Protein Synthesis (E. coli)
- mRNA transcript start codon AUG attaches to the small ribosomal subunit.
- Two different tRNAs are specific to methionine.
- Structural features of the initiating aminoacyl-tRNA are recognized by an initiating factor or protein (IF2), which delivers tRNA to the initiation complex.
- Shine-Dalgarno sequence on the mRNA is complementary to a section of the 16S rRNA and correctly positions the mRNA on the small ribosomal subunit.
Translation Initiation in E. coli
- Initiation involves the binding of different initiation factors (IFs), mRNA, initiator tRNA (with f-Met attached), and the small ribosomal subunit (30S).
- The Shine-Dalgarno sequence on the mRNA positions it on the ribosome.
- The large ribosomal subunit (50S) joins the complex, which releases the initiation factors.
Elongation
- The elongation process links polypeptide chains by transferring amino acid residues from tRNA (in the A site) to the polypeptide chain at the P site on the ribosome.
- This process is facilitated by elongation factors (EFs).
Peptidyl Transferase Reaction
- Peptidyl transferase is an enzyme that catalyzes the formation of peptide bonds between amino acid residues and the polypeptide chain.
Elongation
- Polypeptide synthesis proceeds from N-terminus to C-terminus.
- Chain elongation happens by linking polypeptide chains to incoming tRNA amino acid residues.
- Ribosomes read mRNA in the 5' to 3' direction.
- Translation occurs on polysomes, where multiple ribosomes translate a single mRNA molecule.
- Elongation factors help speed up the elongation process.
Termination
- In E.coli, two release factors (RF1 and RF2) recognize stop codons (UAG and UGA, respectively).
- Water molecules are added to the ribosome, which then hydrolyses the final peptide bond.
- This leads to releasing the polypeptide chain, mRNA, and tRNA and the disassembly of the ribosome complex.
The End Product: Polypeptide
- The end result of protein synthesis is a polypeptide chain (primary structure).
- A chain of amino acids bonded by peptide bonds.
- Primary structure is derived from the DNA base sequence.
- Newly synthesized polypeptide chains are unfolded but rapidly fold into their correct conformation (possibly a stepwise process of side chain fine-tuning).
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Description
Test your knowledge on the crucial processes of protein synthesis, including the roles of aminoacyl-tRNA synthetases and ribosomes. This quiz covers various aspects, from initiation to termination, and the structure of tRNA. Assess your understanding of translation and its mechanisms.
