Transcription and translation Flashcards
What is the role of the promoter region?
Promoters contain sequences that are recognized by RNA polymerase. For example:
Most prokaryotic promoters contain conserved consensus sequences at -10 and -35
RNA polymerase binds to these sequences via the sigma (σ) subunit
AT-rich nature of promoter sequences may facilitate separation of the DNA strands
Note that promoters of eukaryotic genes also have a TA-rich consensus sequence (TATA - the TATA box) at -30.
Consensus sequences are defined by aligning multiple sequences are identifying which bases predominate.
What are the template strand and coding strand?
The DNA strand copied by RNA polymerase is referred to as the template strand. The other strand is referred to as the non-template, partner or coding strand. The term ‘coding strand’ is a little confusing but it is named because it has the same sequence (apart from having T instead of U) as the RNA transcript.
What is the transcription bubble?
RNA synthesis takes place in a ‘transcription bubble’ – ~12-14bp of DNA is transiently separated by RNA polymerase allowing the template strand to be copied. The RNA transcript is elongated by addition to the 3’ end. During transcription elongation RNAP disrupts base-pairing and the transcription bubble moves along the template.
Describe the mechanism of transcription
Nucleotides are added to the 3’ end of the growing RNA chain and therefore RNA synthesis described as occurring in the 5’-to-3’ direction. Note that RNAP is actually moving along the template strand in the 3’-to-5’ direction. Remember the anti-parallel structure of paired nucleic acids.
Give an example of transcriptional termination
Transcription termination takes place via several mechanisms. One mechanism involves a G-C rich hairpin structure forming in the transcript. Following the hairpin there is a U-rich stretch. The hairpin causes RNAP to pause. The U-rich stretch will be weakly paired with As in the DNA template, causing RNA-DNA dissociation and release of RNAP.
What is the open reading frame?
The ‘open reading frame’ (ORF) is the region that codes for a polypeptide. It will start with a start codon and contain a series of codons specifying amino acids uninterrupted by stop codons. The ORF does not take up the entire mRNA. It is flanked by untranslated regions (UTRs) that are not translated but contain important sequences that influence translation and mRNA (amongst other things). The mRNA will start with a 5’ triphosphate and end with a 3’ hydroxyl group.
What is an operon?
In prokaryotes genes coding for proteins that function in the same pathway are often located next to each other and controlled as a single unit that is transcribed into a single polycistronic mRNA. Such a structure is often referred to as an operon.
In the case of polycistronic mRNAs, RNAP will bind to the promoter sequence and generate a transcript that contains multiple open reading frames.
There are some examples of polycistronic mRNAs in eukaryotes but it is not common.
Describe how eukaryotic messenger RNAs undergo specialized processing events at their 5’ and 3’ ends
Addition of a 5’ cap (7-methylguanosine)
addition of 150-200 adenine nucleotides (known as the polyA tail) at the 3’ end
The 5’ cap is a modified guanosine base that is added to the 5’ end early in transcription. It is required for translation and promotes RNA stability and export from the nucleus.
The polyA tail is produced by transcript cleavage followed by the addition of ~200 A residues. It is involved in the process of transcription termination and has roles in RNA stability, RNA export and translation.
How is the genetic code read?
Via transfer (tRNAs) amino acids are attached to specific tRNAs anticodon of the ‘charged’ tRNA base pairs with a complementary codon on the mRNA. Charged tRNAs may be referred to as aminoacyl tRNAs
Describe how tRNAs are charged
Before translation can proceed, the tRNA molecules must be chemically linked to their respective amino acids. This process is often referred to as charging and occurs under the direction of aminoacyl-tRNA synthase enzymes. Aminoacyl-tRNA synthetase enzymes attach amino acids to specific tRNAs. There are 20 amino acids so there are at least 20 tRNAs and enzymes. The charging process requires a high level of accuracy as it is vital that the correct amino acid is attached to the correct tRNA. The charging process requires ATP.
Describe the importance of ribosomes in translation
Charged tRNAs form the link between mRNA and protein
Site of protein synthesis is the ribosome
composed of RNAs + proteins
contains a large and a small subunit
Ribosomal RNAs provide the catalytic function for translation
It is important to note that the catalytic function is provided by the RNA component of the ribosome in the large subunit. The small subunit is involved in ‘decoding’ – pairing charged-tRNAs with the mRNA via anti-codon:codon recognition.
What are the 3 main sites of translation?
A is the aminoacyl site and is where a new charged tRNA will enter the ribosome dependent on the codon present. The anticodon will pair with the codon here. The P site is the peptidyl site and is where the growing polypeptide chain will be located. The E site is the exit where tRNAs will be released when the bond linking tRNA to amino acid is broken. Note that the small subunit is involved in ‘decoding’ whereas the large subunit is the site of peptide bond formation.
Describe translation initiation
In prokaryotes a sequence in the 5’ untranslated region, the Shine-Dalgarno, pairs with the 16S rRNA and places the AUG in the P site of the ribosome
A modified version of methionine, N-formylmethionine, is used for the initiating amino acid.
Initiation also requires protein initiation factors and energy in the form of GTP hydrolysis.
Even though there may be many AUG triplets within a mRNA, the anchoring effect of the Shine-Dalgarno sequence with the 16S rRNA ensures that the correct AUG is in the P site.
Initiation involves the small subunit initially with the large subunit joining to complete the initiation process.
In eukaryotes the small subunit and initiating tRNAi-Met binds to the 5’cap and scans the mRNA until the first AUG is reached
Describe translation elongation
In the elongation process, charged tRNAs will enter the A site pairing codon with anticodon. This is facilitated by translation elongation protein factors, which act as transporters to bring in the aminoacyl tRNA . Once present at the A site, a peptidyl transferase enzyme present in the large subunit will catalyze peptide bond formation between the amino acids at the P and A site. At the same time the bond linking the amino acid and tRNA at the P site is broken, transferring the growing polypeptide chain onto the tRNA at the A site.
Before a new cycle of elongation can take place, the mRNA shifts by 3 bases. This results in the uncharged tRNA being released via the E site, the growing polypeptide chain entering the P site and hence freeing up the A site. This shifting (or translocation) requires translation elongation factors and is driven by energy released by the hydrolysis of GTP.
Describe translation termination
Translation termination occurs when a stop codon (also known as termination or non-sense codon) enters the A site. There are no tRNAs with anti-codons that match the stop codons. However proteins known as release factors can recognize a stop codon at an A site. The release factors enter the A site and stimulate hydrolysis (breakage) of the bond that links the polypeptide chain with the tRNA at the P site.
Polypeptide is released, ribosomal subunits dissociate and mRNA and tRNA are released.
