1. Gene expression, Protein Synthesis and RNA processing Course Title: Gene function and Regulation Course code: BTE-513 MS Biotechnology BRAC University By Dr. M. Mahboob Hossain

2. GENE EXPRESSION The process of gene expression: Information stored in the nucleotide sequences of genes is translated into amino acid sequences of proteins through unstable intermediates called messenger RNAs. Transcription in prokaryotes: Initiation of RNA chains involves three steps: (1) binding of the RNA polymerase holoenzyme to a promoter region in DNA; (2) the localized unwinding of the two strands of DNA by RNA polymerase by, providing a template strand free to base pair with incoming ribonucleotides; (3) the formation of phosphodiester bond between the first few nucleotides in the nascent RNA chain. The holoenzyme remains bound at the promoter region during the synthesis of first eight or nine bonds; then the sigma factor is released and the core enzyme begins elongation phase of RNA synthesis.

5. Five different types of RNA, each encoded by different genes: 1. mRNA:Messenger RNA, encodes the amino acid sequence of a polypeptide. 2. tRNA: Transfer RNA, transports amino acids to ribosomes during translation. 3. rRNA: Ribosomal RNA, forms complexes called ribosomes with protein, the structure on which mRNA is translated. 4. SnRNA: Small nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing (e.g., exon splicing and intron removal). 5. miRNA/siRNA: Micro RNA/small interfering RNA, short ~22 nt RNA sequences that bind to 3’ UTR target mRNAs and result in silencing.

7. Elongation of RNA chain: The covalent extension of RNA chains takes place within the transcription bubble, a locally unwound segment of DNA. The RNA polymerase contains both DNA unwinding and DNA rewinding activities. RNA polymerase continuously unwinds the DNA double helix ahead of the polymerization site as it moves along the double helix. Termination of RNA chain : Termination of RNA polymerase encounters a termination signal. When it does transcription complex dissociates, releasing the nascent RNA molecule.

9. Step 1-Initiation, E. coli model: Fig. 5.3 Each gene has three regions 5’ Promoter, attracts RNA polymerase -10 bp 5’-TATAAT-3’ -35 bp 5’-TTGACA-3’ Transcribed sequence (transcript) or RNA coding sequence 3’ Terminator, signals the stop point

10. Concurrent Transcription, Translation and mRNA degradation: In prokaryotes, the translation and degradation of an mRNA molecule often begin before its synthesis (transcription) is complete. Since mRNA molecules are synthesized, translated and degraded in the 5' to 3' direction, all three process occur simultaneously on the same RNA molecule. In prokaryotes, the polypeptide synthesizing machinery is not separated by a nuclear envelope from the site of mRNA synthesis. Therefore, once 5' end of an mRNA has been synthesized it can immediately be used as template for polypeptide synthesis.

11. EUKARYOTIC GENE EXPRESSION A single RNA polymerase catalyzes all transcription in E. coli, but in eukaryotes there are three RNA polymerases I, II, and III, are more complex, with 10 or more subunits, than the E. coli RNA polymerase. Moreover, unlike the E. coli enzyme, all eucaryotic RNA polymerases require the assistance of ncr proteins called transcription factors in order to initiate synthesis of RNA chains. RNA polymerase I is located in he nucleolus, where rRNAs are synthesized and combined with ribosomal proteins. RNA polymerase I catalyzes the synthesis of all ribosomal RNAs except c small 5S rRNA. RNA polymerase II transcribes nuclear ines that encode proteins and perhaps other genes specifying RNAs. RNA polymerase III catalyzes the synthesis of the isfer RNA molecules, the 5S rRNA molecules, and small ear RNAs.

12. In eukaryotic cells, transcription takes place in the nucleus. The mRNA must be completely synthesized and move thorough the nuclear membrane to the cytoplasm before translation can begin. In eukaryotic cells the regions of genes that code for proteins are often interrupted by noncoding DNA. Thus eukaryotic genes are composed exons, the regions of DNA expressed, and introns, the intervening regions of DNA that do not encode proteins. In addition the RNA undergoes processing before it leaves the nucleus. The long RNA is processed by ribozymes, which removes the intron-derived-RNA amd splice together the exon-derived RNA, producing an mRNA.

13. Initiation mRNA synthesis Unlike their prokaryotic counterparts, eukaryotic RNA polymerases cannot initiate transcription by themselves. All three eukaryotic RNA polymerase require the assistance of protein transcription factors to start the synthesis of an RNA chain. these transcription factors must bind to a region in DNA and form an appropriate initiation fore RNA polymerase will bind and initiate transcri ferent promoters and transcription factors are utilhi RNA polymerases I, II, and III The initiation transcription involves formation of locally unwound segment of DNA providing a DNA strand that is free to function as a template for the synthesis of a complementary strand of RNA. The formation of the locally unwound segment of DNA required to initiate transcription involves the interactions of several transcription factors with specific sequences in the promoter for the transcription unit.

15. RNA chain elongation and the addition of 5' methyl guanosine cap Once eukaryotic RNA polymerases have been released from their initiation complexes, they catalyze RNA chain elongation by the same mechanism as the RNA polymerases of prokaryotes. Early in the elongation process, the 5' ends of eukaryotic pre-mRNAs are modified by the addition of 7-methyl guanosine (7-MG) caps. These 7-MGcaps are added when the growing RNA chains are only about 30 nucleotides long. Termination by chain cleavage and the addition of 3' Poly (A) tails: Transcription proceeds beyond the site that will become the 3' terminus, and the distal segment is removed by endonucleolytic cleavage. This cleavage event produces the 3' end of a transcript usually occurs at a site 11 to 30 nucleotides downstream from a conserved sequence, consensus AAUAAA, and upstream from a G-U rich sequence located near the end of the transcription unit. After the cleavage, the enzyme poly(A) polymerase adds poly (A) tails, tracts of adenosine monophosphate residues about 200 nucleotides long, to the 3'ends of the transcripts.

16. After 8-9 bp of RNA synthesis occurs, sigma factor is released and recycled for other reactions. RNA polymerase completes the transcription at 30-50 bp/second. DNA untwists rapidly, and re-anneals behind the enzyme. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is displaced as the helix reforms.

19. Termination by Chain Cleavage and Addition of 3’ Poly(A) Tails The 3’ ends of RNA transcripts synthesized by RNA polymerase II are produced by endonucleolytic cleavage of the primarv transcripts rather than by the termination of transcription. The actual transcription termination events often occur at multiple sites that are located 1000 to 2000 nucleotides downstream from the site that will become the 3’ end of the mature transcript. That is, transcription proceed beyond the site that will become the 3’ terminus, and the distal segment removed by endonucleolytic cleavage. Poly(A) tails are added to the 3’ ends of transcripts by an enzyme poly(A) polymerase. The 3’ end substrates for poly(A) polye are produced by endonucleolytic cleavage of the transcript downstream from a polyadenylation signal, which has the consensus sequence AAUAAA. 11 to 30 nucleotides downstream from a conserved sequence, consensus AAUAAA, and upstream from a G-U-rich sequence located near the end of the transcription unit. After cleavage, the enzyme poly(A) polymerase adds poly(A) tails, tracts of adenosine monophosphate residues about 200 nucleotides long, to the 3’ ends of the transcripts.

20. The formation of poly(A) tails on transcripts requires a specificity component that recognizes and binds to the AAUAAA sequence, a stimulatory factor that binds to the G-U-rich sequence, an endonuclease, and the poly(A) polymerase. These proteins form a multimeric complex that carries out both the cleavage and the polyadenylation in tightly coupled reactions. The poly(A) tails of eukaryotic mRNAs enhance their stability and play an important role in their transport from the nucleus to the cytoplasm. In contrast to RNA polymerase II, both RNA polymerase I and III respond to discrete termination signals. RNA polymerase I terminates transcription in response to an 18- nucleotide-long sequence that is recognized by an associated terminator protein. RNA polymerase III reponds to a termination signal that is similar to the rho-independent terminator in E. coli.

21. Termination Two types of terminator sequences occur in prokaryotes: Type I (  -independent Palindromic, inverse repeat forms a hairpin loop and is believed to physically destabilize the DNA-RNA hybrid. 3-Termination Type II (  -dependent) Involves  factor proteins, believed to break the hydrogen bonds between the template DNA and RNA.

22. RNA Editing: Altering the Information Content of mRNA Molecules According to the central dogma of molecular biology; genetic information flows from DNA to RNA to protein during gene expression. Normally, the genetic information is not altered in the mRNA intermediary. However, the discovery of RNA editing has shown that exceptions do occur. RNA editing processes alter the information content of gene transcripts in two ways: (1) by changing the structures of individual bases, and (2) by inserting or deleting uridine monophosphate residues. The first type of RNA editing, which results in the substitution of one base for another base, is rare. This type of editing was discovered in studies of the apolipoprotein-B (apo-B) genes and mRNAs in rabbits and humans. Apolipoproteins are blood proteins that transport certain types of fat molecules in the circulatory system. In the liver, the apo-B mRNA encodes a large protein 4563 amino acids long. In the intestine, the apo-B mRNA directs the synthesis of a protein only 2153 amino acids long. Here, a C residue in the pre-mRNA is converted to a U. generating an internal UAA translation-termination codon. which results in the truncated apolipoprotein. UAA is one of three codons that terminates polypeptide chains during translation. If a UAA codon is produced within the coding region of an mRNA, it will prematurely terminate the polypeptide during translation, yielding an incomplete gene prodmct.

25. TRANSLATION AND THE GENETIC CODE The translation of sequence of nucleotide in an mRNA molecule is translated into the appropriate amino acid sequence according to the dictations of the genetic code (each amino acid is specified by one or more codons and each codons contains three nucleotides) can be divided into three stages: (1) polypeptide chain initiation, (2) polypeptide chain elongation, and (3) chain termination. The initiation of translation include all events that precede the formation of a peptide bond between the first two amino acids of the new polypeptide chain. In E. coli, the initiation process involves the 30S subunit of the ribosome, a special initiator tRNA, an mRNA molecule, three soluble protein initiation factors: IF-1, IF-2, and IF-3, and one molecule of GTP. The synthesis of polypeptides is initiated by a special tRNA, designated tRNAf in response to translation initiation codon AUG. The methionine on the initiator tRNAfMet has the amino O ║ group blocked with a formyl (--C—H) group. A distinct methionine tRNA, tRNAfMet, responds to internal methionine codons. Both methionine tRNAs have the same anticodon, and both respond to the same codon (AUG) for methionine. However, only

26. methionyl- tRNAfMet interacts with protein factor IF- 2 to begin initiation process. Polypeptide initiation begins with the formation of two complexes : (1) one contain initiation factor IF-2 and metyhionyl-tRNAfMet, and (2) the other contains an mRNA molecule, 30S ribosomal subunit and initiation factor IF-3. The IF-2/metyhionyl-tRNAfMet complex and the mRNA/30S subunit/IF-3 complex subsequently combine with each other and with intiation factor IF-1 and one molecule of GTP to form the complete 30S initiation complex.

27. The final step in the initiation of translation is the addition of 50S subunit to the 30S initiation complex to produce the 70S ribsome. Initiation factor IF-3 must be released from the complex before the 50S subunit can join the complex. Metyhionyl-tRNAfMet is the only aminoacyl-tRNA that can enter the P-site directly, without first passing through the aminoacyl A site. With the initiator AUG positioned in the P site, the second codon of the is in register with A site, dictating the aminoacyl-tRNA binding specificity at the site and setting the stage for the second phase in polypeptide synthesis, chain elongation. The initiation of translation is more complex in eukaryotes, involving several soluble initiation factors. Nevertheless, the overall process is similar except for two features. (1) The amino group of the methionine on the initiator tRNA is not formylated as in prokaryotes. (2) The initiation complex forms at the 5' terminus of the mRNA, not at the Shine- Dalgarno/AUG translation start site as in E. coli. In eukaryotes, translation frequently begins at the AUG closest to the 5' terminus of the mRNA molecule.

28. In eukaryotes, a cap-binding protein (CBP) binds to the 7-methyl guanosine cap at 5' terminus of the mRNA. Then other initiation factors bind to the CBP-mRNA complex followed by the small (40S) subunit of ribosome. The entire initiation complex moves 5' →3' along the mRNA molecule searching for an AUG codon. When an AUG codon is found, the initiation factors dissociate from the complex and the large (60S) subunit of ribosome binds to the methionyl-tRNA/mRNA/40S subunit complex, forming the complete (80S) ribosome. The 80S ribosome/tRNA/mRNA complex is ready to begin the second phase of translation, chain elongation.

30. Peptide elongation The process of polypeptide chain elongation is basically the same in both prokaryotes and eukaryotes. The addition of each amino acid to the growing polypeptide occurs in three steps: (1) binding of an aminoacyl-tRNA to the A site of the ribosome. (2) transfer of the growing polypeptide chain from the tRNA in the P site to the tRNA in the A site by the formation of new peptide bond, and (3) translocation of the ribosome along the mRNA to position the next codon to A site. In the first step an aminoacyl-tRNA enters and become bound to the A site of the ribosome, with specificity provided by the mRNA codon in register with A site. This step requires elongation factor Tu carrying a molecule of GTP (EF- Tu.GTP). The second step in chain elongation is the formation of a peptide bond between the amino group of the amino acyl-tRNA in the A site and the carboxyl terminus of the growing polypeptide chain attached to the tRNA at the P site. This key reaction is catalyzed by the enzyme peptidyl transferase. During the third step in the chain elongation, the peptidyl-tRNA present in the A site of the ribosome is translocated to the P site, the uncharged tRNA in the P site is translocated to the E site, as the ribosome moves three molecules toward the 3' end of the mRNA molecule. The translocation step requires GTP and elongation factor G (EF-G).

31. Polypeptide chain elongation undergoes termination when any of three chain termination codons (UAA, UAG and UGA) enters the A site of the ribosome. These three stop codons are recognized by soluble proteins called release factors (RFs). In E. coli there are two release factors RF-1 and RF-2. The RF-1 recognizes UAA and UAG; RF-2 recognizes UAA and UGA. In eukaryotes single release factor (eRF) recognizes all three termination codons. When the tRNA that recognizes the second codon moves into position on the ribosome, the first amino acid is transferred by the ribosome. After the ribosome joins the two amino acids with a peptide bond, the first tRNA molecule leaves the ribosome. The ribosome then moves along the mRNA to the next codon. As the proper amino acids are brought into line one by one, peptides bonds are formed between them, and a polypeptide chains results. Translation ends when one of three nonsense codons in the mRNA is reached. When the ribosome arrives at this codon, it comes apart into its two subunits, and the mRNA and newly synthesized polypeptide chains are released. The ribosome, the mRNA and the tRNAs are then available to be used again