SIJ1003 Biochemistry of Cell - Week 12 Lectures (PDF)

12/31/24   SIJ1003 Biochemistry of Cell Week 12 Lectures 1 31 DEC 2024 1    12/31/24   ANIMATIONS: TRANSCRIPTION: https://www.youtube....

12/31/24   SIJ1003 Biochemistry of Cell Week 12 Lectures 1 31 DEC 2024 1    12/31/24   ANIMATIONS: TRANSCRIPTION: https://www.youtube.com/watch?v=ztnfrVHDJbQ CENTRAL DOGMA: Flow of Genetic Information 4 2    12/31/24   Protein Synthesis 5 Coding/sense strand/non-template strand 3    12/31/24   Transcription 7 Transcription in prokaryotes } Transcription Unit extends from the promoter to the terminator: Core enzyme: } made up of four peptide subunits (2 α, 1β & 1β’) } responsible for 5’→3’ RNA polymerase activity } lacks specificity (can not recognize the promoter region on the DNA template) σ subunit: } σ subunit (‘σ factor’) enables polymerase to recognize promoter region on DNA } σ factor plus the core enzyme compose Holoenzyme 4    12/31/24   Transcription in prokaryotes Properties of prokaryotic RNA polymerase: } Multi-subunit enzyme } recognizes nucleotide sequences (Promoter region) at the beginning of a stretch of DNA to be transcribed } makes a complementary RNA copy of DNA template } recognizes the end of DNA sequence to be transcribed (Terminator region) Steps in Transcription Initiation Promoter Transcription unit Involves the binding of RNA polymerase to a 5ʹ′ 3ʹ′ } 3ʹ′ 5ʹ′ Start point promoter region on DNA RNA polymerase 1 Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and } Characteristic nucleotide sequences of the the polymerase initiates RNA synthesis at the start point on the template strand. promoter region recognized by RNA polymerase 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ are: Unwound DNA RNA Template strand of DNA transcript } Pribnow box: Stretch of seven nucleotides centered around ten nucleotides to the left of the initial base of mRNA. } TGTTG sequence: located about 35 nucleotides to the left of initial base of mRNA. 5    12/31/24   mRNA - Prokaryotes TGTTG sequence Pribnow box Transcription start site Steps in Transcription Elongation } After recognition of the promoter region, RNA polymerase begins to synthesize a transcript of DNA sequence. } Unlike DNA polymerase, RNA polymerase Ø does not require a primer. Ø has no known endonuclease or exonuclease activity. Ø does not require unwinding enzymes or helix destabilizing enzymes. } Binding of enzyme to DNA template results in a local unwinding of DNA helix. } Like DNA polymerase, RNA polymerase uses nucleoside triphosphates & releases pyrophosphate each time a nucleotide is added. 6    12/31/24   Steps in Transcription Elongation Promoter Transcription unit } RNA is synthesized 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ Start point Ø from its 5’ end to 3’ end 1 Initiation. After RNA polymerase binds to RNA polymerase the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the Ø antiparallel to DNA template start point on the template strand. 5ʹ′ 3ʹ′ Ø template is copied similar to as in 3ʹ′ 5ʹ′ Unwound RNA Template strand of DNA DNA synthesis (‘G’ on DNA specifies DNA transcript 2 Elongation. The polymerase moves downstream, unwinding the ‘C’ in RNA; ‘C’ specifies a ‘G’; ‘T’ DNA and elongating the RNA transcript 5ʹ′ → 3 ʹ′. In the wake of transcription, the DNA strands re-form a double helix. Rewound specifies an ‘A’ but ‘A’ specifies a ‘U’ RNA instead of a ‘T’ 5ʹ′ 3ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ 5ʹ′ RNA } Elongation of RNA continues until a transcript termination signal is reached. Steps in Transcription Promoter Transcription unit Termination 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ DNA } RNA polymerase recognizes termination Start point RNA polymerase 1 Initiation. After RNA polymerase binds to regions on DNA template (ρ - independent the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the termination) start point on the template strand. 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ } ρ-factor (rho) may be required for the Unwound RNA Template strand of DNA release of RNA & RNA polymerase (ρ - DNA transcript 2 Elongation. The polymerase moves downstream, unwinding the dependent termination) Rewound DNA and elongating the RNA transcript 5ʹ′ → 3 ʹ′. In the wake of transcription, the DNA strands re-form a double helix. } Termination region exhibits two- fold RNA 5ʹ′ 3ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ symmetry owing to the presence of RNA 5ʹ′ palindromes. transcript 3 Termination. Eventually, the RNA transcript is released, and the } RNA transcript of DNA palindrome form a polymerase detaches from the DNA. stable hairpin turn that slows down the 5ʹ′ 3ʹ′ 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ movement of RNA polymerase at this site. Completed RNA transcript 7    12/31/24   3’~CGACTGNNNCAGTCG~5’ DNA 5’~GCTGACNNNGTCAGC~3’ Transcription RNA 5’~GCUGACNNNGUCAGC~3’ G ≡≡≡ C C ≡≡≡ G U === A G ≡≡≡ C A === U C ≡≡≡ G NNN RNA hairpin Example of palindrome: Each of two strands has the same sequence when read in the same direction (5’→3’) Steps in RNA biosynthesis Promoter Terminator 5’ 3’ 3’ 5’ Sigma factor Sigma factor recognizes promoter region 5’ 3’ 3’ 5’ Core enzyme Core enzyme binds & begins to synthesize RNA 8    12/31/24   5’ 3’ 3’ 5’ Rho factor Elongation continues until termination region is reached 5’ 3’ 3’ 5’ Rho factor terminates transcription 3’ 5’ Newly synthesized RNA 9    12/31/24   Transcription in Eukaryotes Eukaryotic Promoter region } TATA box or Hogness box: A consensus sequence of nucleotides (almost identical to that of Pribnow box) centered about 25 nucleotides to the left of the initial base of the DNA. } CAAT box: A consensus sequence of nucleotides centered between 70 & 80 nucleotides to the left of the initial base of the DNA. } One or both of these sequences serve as recognition sites in eukaryotic promoter. 10    12/31/24   mRNA - Eukaryotes 40 bases 25 bases CAAT box TATA box Start of (−70 sequence) (Hogness box) Transcription (−25 sequence) DNA double helix Eukaryotic gene promoter region Transcription in Eukaryotes } Three distinct species of RNA polymerase are found in eukaryotic cells. } RNA polymerase I synthesizes the large ribosomal RNAs (rRNAs)in the nucleolus. } RNA polymerase II synthesizes the precursors of messenger RNAs (mRNAs) that will be translated to produce proteins. } RNA polymerase III produces small RNAs, including transfer RNAs (tRNA) & small (5s) ribosomal RNAs (rRNAs). 11    12/31/24   Post modification to RNA Eukaryotic messenger RNA } RNA (primary transcript, sometimes called as “hn RNA” for heterogeneous nuclear RNA) synthesized by RNA polymerase contains sequences present in cytoplasmic mRNA. The primary transcript is extensively modified after transcription. These modifications are: 1. 5’ “capping” } First of the processing reactions for hn RNA. } Cap is 7-methyl guanosine attached through a triphosphate linkage to the 5’ end of mRNA (enzymatic reactions). } Appears to facilitate the initiation of translation & helps stabilize mRNA. Post modification to RNA 2. Addition of a poly (A) tail } present in most eukaryotic mRNAs } a chain of 40 – 200 adenine (A) nucleotides attached to the 3’ end. } added after transcription by the nuclear enzyme poly (A) polymerase. } may help stabilize mRNA & facilitate its exit from the nucleus. } upon entry into cytoplasm, tail is gradually shortened. 12    12/31/24   5’end 3’ end 7-methyl Gppp pA.pA.…pA- OH Post modification to RNA 3. Removal of introns } Maturation of eukaryotic mRNA involves the removal of RNA sequences (introns or intervening sequences) from the primary transcript that do not code for protein. } Small nuclear RNAs (Sn RNAs) facilitate the splicing of some exons by forming base pair with each end of intron, thus bringing neighboring exons in correct alignment for enzymatic splicing, resulting in excision of introns. } Now mature mRNA leaves the nucleus through nuclear pores. 13    12/31/24   Exon1 Intron Exon 2 Excision of intron & splicing of exons in eukaryotic RNA RNA processing or RNA splicing 28 14    12/31/24   29 Prokaryotes vs Eukaryotes Prokaryotes Eukaryotes – no nucleus to separate - transcription occurs in nucleus transcription and translation and translation in cytoplasm – Naked DNA - Nucleosome/ chromatin – translation can begin - pre-mRNA produced, then immediately while mRNA is processed to produce mature being transcribed mRNA - Mature mRNA exits nucleus into the cytoplasm to be translated. 15    12/31/24   Google list } Promoter region in eukaryotes vs prokaryotes 16