1- In E. coli protein synthesis, the initiation complex contains A.50S ribosomal
ID: 302925 • Letter: 1
Question
1- In E. coli protein synthesis, the initiation complex contains
A.50S ribosomal subunit, 23S RNA, IF1, IF3, mRNA, fMet-tRNA. B.30S ribosomal subunit, 5S and 16S rRNA, IF-1, IF-2, IF-3, GTP, mRNA, fMet-tRNA.
C.50S ribosomal subunit, 16S RNA, IF1, IF2, IF3, GTP, mRNA, fMet-tRNA. D.30S ribosomal subunit, 16S RNA, IF1, IF2, IF3, GTP, mRNA, fMet-tRNA.
E.30S ribosomal subunit, 23S rRNA, IF1, IF2, IF3, GTP, mRNA, fMet-tRNA.
2- In CRISPR/Cas9 genetic engineering, the guide RNA (gRNA)
A.deactivates the enzymatic activity of Cas9. B.All of the answers are correct. C.always recognizes the same target sequence in DNA
D.is made by joining crRNA with a linker ssRNA E.binds to the target DNA
Explanation / Answer
1. The 5’ untranslated region of mRNA has the binding site for the ribosome. In prokaryotes, it is the shine Dalgarno’s sequence. First, there is a formation of a complex on the mRNA during translation of mRNA. First, three initiation factor proteins IF1, IF2, and IF3 bind to the small subunit of the ribosome to form the pre-initiation complex. This complex along with formyl methionine carrying tRNA (fmet-tRNA) bind to mRNA to form initiation complex. This is followed by the large subunit binding to the complex to form functional ribosome. The initiation factors are released from the complex.
The entire process occurs as follows:
i) The initiation factor 3 binds to 30S ribosomal subunit. The 30S subunit in E.coli consist of 16S rRNA while 50S subunit has 5S and 23S rRNA.
ii) The mRNA binds to 30S ribosomal subunit. A base pair is formed between the Shine Dalgarno’s sequences to the complementary sequence present near the 3? end of the 16S rRNA. The mRNA binding occurs such that AUG codon lies on the peptidyl (P) site. The second codon will now lie on aminoacyl (A) site.
iii) The tRNA for formylated methionine (FMet–tRNAFMet) attaches to P-site by initiation factor 2, with utilization of GTP. IF-1 will prevent the binding of FMet–tRNAFMet to the A-site.
iv) The initiation factors 3, 2 and 1 are released and 50S ribosomal unit binds to 30S. Thus, a 70S ribosome is formed.
Right option is D. The initiation complex contains 30S ribosomal subunit, 16S rRNA, IF 1-3, mRNA and fmet tRNA. 50S subunit (containing 23S rRNA and 5S rRNA binds to this initiation complex to form the 70S ribosome (after release of IF).
2. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its associated protein 9 nuclease (Cas9) are the new technology employed for gene editing. Three types of CRISPR mechanism have been identified, of which Type II is the most studied mechanism. In Type II mechanism, only Cas9 is required for gene silencing. Cas9 processes the crRNAs and destroys the target DNA. Cas9 has six domains: REC I, REC II, Bridge Helix, PAM Interacting, HNH, and RuvC. The Rec1 domain binds to guide RNA. Bridge helix is arginine rich and initiates the cleavage after binding of target DNA. PAM-interacting domain (protospacer adjacent motif) confers specificity of PAM for initial binding to target DNA. HNH and RuvC domains are nuclease domains for cleavage of DNA.
In absence of guide RNA, Cas9 is inactive. When the target gene is engineered with CRISPR, guide RNA is present that is single stranded forming a T-Shape. The T-shape has one tetraloop and two or three stem loops. This guide RNA has a 5? end complimentary to target gene sequence. This artificial guide DNA binds to the Cas9 protein and causes a conformation change in the protein. As a result, Cas9 is activated and searches the target DNA sequences complementary PAM sequences. PAM is 2-3 amino acid sequences on target DNA present within one nucleotide downstream of the complementary region to guide RNA. When Guide RNA finds the target sequence with its appropriate PAM sequences, Cas9 will melt the bases upstream to the PAM sequence. These guide RNA sequences will now bind the complementary sequences on target DNA in presence of Cas9. The target DNA is cut after the third nucleotide upstream of PAM by RUVC and HNH domains. Hence, gene silencing will occur, altering gene expression.
Hence, guide RNA will activate Cas9 enzymatic activity. Guide RNA is composed of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA; acts as a scaffold). They are around 100 nucleotide long. The 20 nucleotides at its end hybridize with its target sequences while the rest is required for CAS binding and activity. The target sequences that are recognized by guide DNA depends on which targets needs to be deleted. Complementarity determines the binding of guide RNA to target sequences. Hence, the guide RNA is designed to be complementary to target DNA sequences. Guide RNAs do not recognize the target sequences. Cas protein is required to melt the DNA before the guide RNA binds to the DNA target.
Right answer is E. guide RNA binds to target DNA.
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