mic270 chapter 4.pdf

MIC270 BASIC MOLECULAR BIOLOGY CHAPTER 4 Gene Expression Learning Outcomes At the end of this topic, the student should be able to: Identify the process involve in gene expression including replication, transcription, splicing and translation. Differentiate between...

MIC270 BASIC MOLECULAR BIOLOGY CHAPTER 4 Gene Expression Learning Outcomes At the end of this topic, the student should be able to: Identify the process involve in gene expression including replication, transcription, splicing and translation. Differentiate between expression of gene in eukaryote and prokaryote Gene Regulation Gene regulation process used to control the timing, location and amount in which genes are expressed. They involved regulatory proteins Capable of binding to a cell’s DNA near the promoter of the genes Control gene expression in some circumstances but not in others Positive regulation binding of regulatory proteins makes it easier for an RNA polymerase to initiate transcription (bind -> transcription) Negative regulation binding of the regulatory proteins prevents transcription from occurring (bind -> stop transcription) Differences between prokaryotes and eukaryotes: Prokaryote gene expression typically is regulated by an operon, the collection of controlling sites adjacent to polycistronic protein- coding sequences. Eukaryotic Eukaryotic genes also are regulated in units of protein-coding sequences and adjacent controlling sites, but operons are not known to occur. Eukaryotic gene regulation is more complex because eukaryotes possess a nucleus. (transcription and translation are not coupled). Two “categories” of eukaryotic gene regulation exist: Short-term - genes are quickly turned on or off in response to the environment and demands of the cell. Long-term - genes for development and differentiation.  DNA REPLICATION When?  S (synthesis) phase of cell cycle  Creates copy of DNA and two copies are held together by centromere  Thousands of times per second throughout the body Where?  Always in the nucleus of eukaryotic cells.  All cells that are not in G0 phase (no longer dividing). Why?  When cells divide through meiosis or mitosis, new cells need their own DNA How?  Two different mechanisms: lagging strand and leading strand, but always in the 5’ to 3’ direction.  Eukaryotic chromosomes average about 150 million nucleotides, so multiple replication forks are needed to finish the job.  Semi-conservative Key enzymes 1. Topoisomerases unknot the DNA. 2. Helicase unzips the DNA 3. RNA primase adds RNAprimer 4. DNA polymerase adds nucleotides, repairs mismatch pairs, and removes the RNA primer 5. DNA ligase seals the okazaki fragments together by creating phosphodiester bonds.  RNA primase DNA ligase DNA polymerase Topoisomerase DNA polymerase Helicase Source:wikipedia.com 1 2 3 4 17 ACTIVITY 1 Work in pair or trio On a piece of paper, draw and simulate the process of DNA replication that you have learned just now Identify the enzymes involved in both leading and lagging strand  GENE EXPRESSION  Many Bacterial Genes Are Transcribed and Regulated Together in an Operon  An operon is a cluster of genes sharing a promoter and regulatory sequences.  Genes are transcribed together, so mRNAs are several genes represented on one mRNA (polycistronic).  First example: the lac operon Gene Expression: Prokaryotes Operon – grouped genes that are transcribed together – code for functionally similar proteins Key Players  Promoter – section of DNA where RNA polymerasebinds  Operator – Controls activation of transcription  on off switch  between promoter and genes for proteins – structural genes  Repressor protein – binds to operator to block RNA polymerase and shut down transcription  Turns off the operon  Corepressor – keeps the repressor protein on the operator  Trp operon  Inducer – pulls repressor off the operator  Turns on the operon – lactose on the lac operon  Regulatory gene – produces the repressor protein  Structural genes – code for proteins Gene Expression: Eukaryotes Eukaryote gene expression is regulated at six levels: 1. Transcription 2. RNA processing 3. mRNA transport 4. mRNA translation 5. mRNA degradation 6. Protein degradation Fig. 18.1 mRNA protein Anti codon ACTIVITY 2: Watch this video! Transcription and Translation From DNA to RNA to protein Genes in DNA contain information to make proteins. The cell makes mRNA copies of genes that are needed. The mRNA is read at the ribosomes in the rough ER. Protein is produced. Transcription RNA polymerase is the enzyme responsible for making mRNA copies of genes. DNA unzips at the site of the gene that is needed. Transcription RNA polymerase matches bases in the sense strand with RNA bases, building a strand of mRNA that carries the information encoded in the DNA. Transcription Encoded in DNA is a signal telling RNA polymerase where to stop. Transcription ends at that point. Transcription The completed mRNA molecule then moves from the nucleus to the rough ER for translation. SPLICING Remember that the story is much more complicated in Eukaryotes? Important differences A ‘cap’ is added to the 5’ end of the mRNA A polyA tail is added to the 3’end Introns are removed by a process called splicing Translation  Initiation begins with a tRNA bearing methionine (met) attaching to one of the ribosomal units. The codon for methionine is a universal “start” codon for “reading” the mRNA strand. Translation  The ribosomal unit tRNA binds to mRNA where the code for met is located (AUG). The anticodon (UAC) of the tRNA matches the “start” codon on mRNA (AUG). Small ribosomal subunit Translation  The larger ribosomal subunit now binds to the smaller unit, forming a ribosomal complex. The tRNA binds to the first active site on the ribosome. Translation may now begin. Translation  The second codon in mRNA (GUU) matches the anticodon of a tRNA carrying the amino acid valine (CAA). The second tRNA binds to the second active site on the large subunit. Translation  A catalytic site on the larger subunit binds the two amino acids together using dehydration synthesis, forming a peptide bond between them. Translation  The first tRNA now detaches and goes of to find another met in the cytoplasm. The mRNA chain shifts over one codon, placing the second codon (CAU) over the second active site. Translation  A tRNA with an anticodon (GUA) matching the exposed codon (CAU) moves onto the ribosome. This tRNA carries histidine (his). Translation  A new peptide bond forms between val and his on the catalytic site. The tRNA that carried val will detach and find another val in the cytoplasm. The mRNA strand will then shift over one more codon. Translation  The process continues until the ribosome finds a “stop” codon.  The subunits detach from one another, the mRNA is released  The polypeptide chain moves down the ER for further processing.  The initial met is removed and the chain is folded into its final shape. Summary DNA to protein  Reverse Transcription The transfer of genetic information from RNA to DNA By the enzyme reverse transcriptase found in retrovirus This enzyme make cDNA (complementary DNA) from mRNA and obtain a gene sequence without the introns 4. Reverse transcription – from RNA to DNA END OF LESSON