Lec 3 Outline - 2024 A01 - PDF

CFTR is a chloride ion channel http://i.stack.imgur.com/0WJib.jpg Mutations in the gene lead to non-...

CFTR is a chloride ion channel http://i.stack.imgur.com/0WJib.jpg Mutations in the gene lead to non-functional CFTR No chloride ion transport Improper hydration of airways and intestines http://66.media.tumblr.com/ef8dc4bfe8d73d2315a1baab 3c26a0d4/tumblr_inline_nwdlboBQcT1qf0536_1280.jpg  CFTR is a chloride ion channel -if ion channel functional à ions and fluid enter the “lumen” -if ion channel nonfunctional à no ions or fluid enter the “lumen” “Mini guts” = organoids -functional CFTR can be activated by a chemical named forskolin http://www.news-medical.net/image.axd?picture=2015%2F3%2Famsbiopr142-lowresimage.jpg  CRISPR edited cells https://www.eurekalert.org/multimedia/pub/web/118074_web.jpg CFTR F508Del = mutation in CF patients - These cells taken from the patient’s intestine - Compared to cells that were edited with CRISPR to correct the mutation = F508Del-Corrected clone (S1-c1 and S1-c2) https://asm.org/Articles/Cultures-Magazine/Volume-4,-Issue-4-2017/The-Designer-Baby-Distraction Relevant reading: Morris text, 4th edition, Chapters 4&5  GENETIC INFORMATION IN DNA DIRECTS THE ACTIVITIES IN A CELL The Central Dogma  GENETIC INFORMATION IN DNA DIRECTS THE ACTIVITIES IN A CELL The Central Dogma Exceptions exist. E.g. viruses, prions  RNA vs DNA Ribose and Deoxyribose  RNA vs DNA Uracil and Thymine  Many scientists believe the first nucleic acids were RNA molecules. RNA is involved in many cellular processes, including all the steps of the central dogma. RNA also has enzymatic properties. It is thought that DNA is used by cells because it is more stable than RNA molecules.  RNA vs DNA DNA RNA Sugar Deoxyribose Ribose Bases A, T, C, G A, U, C, G 5′ end Monophosphate Triphosphate Size Very large Smaller Strands Double Single  DNA serves as the template for RNA production by the cell. Although transcription occurs in different places in prokaryotes and eukaryotes, the process is similar in both types of cells. TEMPLATE vs NONTEMPLATE STRANDS  INITIATION AND TERMINATION OF TRANSCRIPTION A DNA molecule usually contains many genes Transcription is initiated at a promoter sequence and ends at a terminator sequence. The transcript is synthesized in a 5'-to-3' direction. RNA polymerase & associated proteins bind to the DNA duplex at the promoter. Terminator Promoter DNA RNA TATA TATA Which strand is transcribed can differ from one gene to the next.  Transcription factors and RNA polymerase bind double-stranded DNA at promoter sequences. Promoter sequences are conserved DNA sequences.  INITIATION AND TERMINATION OF TRANSCRIPTION Many Eukaryotic promoters contain a sequence similar to TATAAA, which is known as a TATA box. The first nucleotide to be transcribed is usually positioned about 25 base pairs from the TATA box. Terminator Promoter DNA RNA TATA TATA Transcription will continue until RNA polymerase encounters a terminator  PROMOTER RECOGNITION IN EUKARYOTES IS COMPLEX Enhancers can be located in, near or some distance from a gene  PROMOTER RECOGNITION IN EUKARYOTES IS COMPLEX A general transcription factor known as TATA-box binding protein (TBP) interacts with the TATA box region of the promoter during transcription initiation.  PROMOTER RECOGNITION IN EUKARYOTES IS COMPLEX Proteins bound to enhancers recruit a mediator complex RNA POLYMERASE II ADDS NUCLEOTIDES This step is called Elongation. POLYMERIZATION REACTION RNA POLYMERASE IN The RNA polymerase PROKARYOTES contains separate channels for (a) the entry of the trinucleotides, (b) DNA to be transcribed, and (c) for the exit for the RNA transcript and (d) transcribed DNA. PRIMARY TRANSCRIPT IN PROKARYOTES The primary transcript is the mRNA. In prokaryotes, the primary transcripts can contain the information from more than one gene, directing synthesis of multiple proteins à polycistronic mRNA. EUKARYOTIC Transcription & Translation occur in PRIMARY separate compartments. TRANSCRIPT Eukaryotic transcripts undergo processing prior to translation. EUKARYOTIC 5′ Cap on Eukaryotic mRNA PRIMARY TRANSCRIPT  POLY(A) TAIL ON EUKARYOTIC MRNA Polyadenylation adds a This modification plays an poly(A) tail to the 3' important role in the export end. of the mRNA to the = ~ 250 A-bearing cytoplasm of the cell. nucleotides Primary Poly(A) tail transcript (RNA) 5' cap EUKARYOTIC Eukaryotic cell PRIMARY TRANSCRIPT Exon Intron DNA 5' cap Poly(A) tail Primary transcript (RNA) The 5' end is modified by Polyadenylation adds a special nucleotide called a poly(A) tail to the the 5' cap. mRNA 3' end. Spliced exons BOTH these modifications contribute to transcript stability.  RNA SPLICING -1 Not every stretch of the primary transcript is translated to protein in eukaryotes Protein coding regions = exons; noncoding regions = introns ~90% of all human genes contain at least 1 intron Exon Intron Primary transcript Spliceosome components The spliceosome is composed of RNA & proteins that catalyze the splicing together of exons and removal of noncoding introns.  RNA SPLICING -2 Exon Intron Primary transcript Spliceosome components A site within the intron attacks the 5’ splice site. RNA SPLICING - 3 The cleaved 5’ splice site attacks the 3’ splice site. Lariat quickly breaks down Spliced exons are adjacent in into individual nucleotides. the processed RNA. ALTERNATIVE SPLICING Note: Other RNA transcripts are processed differently from protein coding RNA transcripts à e.g. tRNAs, rRNAs etc. TRANSLATION AND PROTEIN STRUCTURE Proteins are diverse and versatile Proteins are made up of building blocks called amino acids The exact sequence of amino acids will determine each protein’s shape and function How are proteins made and folded into a specific shape in order to be functional? AMINO ACID STRUCTURE  HYDROPHOBIC AMINO ACIDS Tend to be buried in the interior folds of proteins. HYDROPHILIC AMINO ACIDS SPECIAL AMINO ACIDS Small & flexible Creates kinks Forms bridges within and between proteins = disulfide bonds N C Some nomenclature: Protein = polypeptide Amino acid = residue N C No matter what the function is of a polypeptide, its ability to carry out that function will be dictated by its 3-dimensional shape. AMINO ACID STRUCTURE  PRIMARY STRUCTURE A-M-A-M Ala-Met-Ala-Met (alanine-methionine-alanine-methionine) ALPHA HELIX The bonds occur between the amino acid functional groups, NOT the R groups. The polypeptide chain is twisted tightly in a right- handed coil BETA SHEET