What is a “promoter” in bacteria? Is a promoter composed of DNA, RNA, or protein? What is the relationship between a promoter and sigma factor?

• A promoter is a section of DNA that promotes the start of transcription.
• Transcription begins when sigma, as part of the holoenzyme complex, binds to the promoter

What happens during the “elongation phase” of transcription?

• During elongation, the rudder helps steer the template and non-template strands, the enzyme’s active site catalyzes the addition of nucleotides to the 3’ end of the growing RNA molecule and the zipper helps separate the newly synthesized RNA from the DNA template

How does transcription in bacteria end?

Transcription stops when RNA polymerase transcribes a DNA sequence that functions as a transcription termination signal and as soon as it is synthesized, this portion of RNA folds folds upon itself to form a short double helix that is held together by complementary base pairing and this results in the physical separation of RNA polymerase and the RNA transcript

How does transcription in eukaryotes differ from transcription in prokaryotes?

• Eukaryotes have three polymerases and RNA polymerase II is the only polymerase that transcribes protein-coding genes
• Eukaryotes have more diverse promoters
• TATA box is centered about 30 base pairs upstream of the transcription site
• Instead of sigma, eukaryotes use basal transcription factors which assemble at the promoter and RNA polymerase follows
• Termination of eukaryotic protein-coding genes involves a short sequence called the poly(A) signal and after it is transcribed, the RNA is cut by an enzyme downstream of the poly (A) signal as the polymerase continues to transcribe the DNA template, RNA polymerase eventually falls off and terminates transcription

In eukaryotes there are at least three different RNA polymerase enzymes. Which one is responsible for transcribing protein-coding genes?

• RNA polymerase II

In eukaryotes, how does a mature mRNA differ from a primary transcript? Draw a schematic of a mature mRNA, and explain the function of each part of the molecule.

• The primary transcript is the result of transcription using the DNA template and becomes a mature mRNA once the spliceosome has spliced out introns.

Explain how alternative splicing allows a single gene to code for more than one protein.

• Since the RNA can be spliced in more than one way, this allows the production of different, related mRNAs and proteins from one gene.

What are the minimal components required for protein synthesis?

• Ribosomes, mRNA, amino acids, tRNA, ATP and GTP

Where does protein synthesis occur in bacteria? in eukaryotes?

• In bacteria, protein synthesis occurs concurrently with transcription because there is no nuclear envelope to separate the two processes.
• Multiple ribosomes, polyribosome, attach to each mRNA and many copies of a protein can be produced from one mRNA.
• In eukaryotes, primary transcripts are processed in the nucleus to produce a mature mRNA, which is then exported to the cytoplasm and polyribosomes attach to it and begin synthesizing proteins.

How does tRNA serve as an “adapter” in protein synthesis? What is the relationship between a codon and an anticodon?

• tRNA serves as an “adapter” by acting as an interpreter and allowing amino acids to interact with an mRNA template.

What is the relationship between a codon and an anticodon? How can 40 tRNAs translate 61 different codons?

• The anticodon forms complementary base pairs with an mRNA codon.
• According to the wobble hypothesis, particular non-stranded base pairs, such as G-U, are acceptable in the third position of a codon and do not change the amino acid that the codon specifies therefore the wobble in the third position of a codon allows just 40 or so tRNAs to bind to all 61 mRNA codons.

What do aminoacyl tRNA synthetases do?

• Aminoacyl tRNA synthetases catalyze the addition of amino acids to tRNAs.
• There is a different one for each 20 amino acids and one or more tRNAs, there is a binding site for a particular amino acid and tRNA

What would be the effect if a mutation in the gene for a particular aminoacyl tRNA synthetase caused the enzyme to become completely non-functional?

How do mRNA, tRNA, and ribosome subunits come together to start the process of translation in bacteria?

• The process begins when a section of rRNA in a small ribosomal subunit binds to complementary sequence on an mRNA, the mRNA region is called the ribosomal binding site.
• Initiator aminoacyl tRNA binds to start codon and it is followed by the binding of the large subunit after which translation can begin.

Describe the sequence of events that occurs during translation as a protein elongates by one amino acid and the ribosome moves down the mRNA. Does protein or RNA catalyze protein synthesis?

• The translation of each mRNA codon begins when an aminoacyl tRNA diffuses into the A site and stays if its anticodon matches a codon in the mRNA.
• A peptide bond then forms between the amino acid held by the aminoacyl tRNA in the A site and the growing polypeptide, which was held by a tRNA in the P site.
• The ribosome moves down the mRNA by one codon, and all three tRNAs move down one position and the tRNA in the E site exits.
• Protein synthesis is catalyzed by RNA.

How does protein synthesis conclude when the ribosome reaches the end of the message?

• Release factors recognize stop codon and fill the A site although they do not carry an amino acid.
• Then the protein’s active site catalyzes the hydrolysis of the bond that links tRNA in the P site to the polypeptide chain.
• Finally, the polypeptide chain is released from the ribosome, the ribosome separates from the mRNA and the two ribosomal subunits dissociate.