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Notes for Second Midterm.docx

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George S Espie

Notes for Second Midterm  Folding is important since it reduces the loss of function, prevents diseases which are associated with miss-folded proteins. -helix – it is right handed helix stabilized by H bonds between C=O of peptide group n and N- H of peptide group n+4.  3.6 AA are required for one complete turn. The R group points outwards.  Ala, Leu, Met, Glu (Normally found in  helix).  Pro, Gly, Ser, Thr (Normally not found in  helix and are referred to as helix breaker)  Hydrogen bond is between the O at carbonyl group to the H atom on the amid group on the n+4 amino acid. -sheet – adjacent chains held together by hydrogen bonds between backbone. Stability depends on the number of hydrogen bonds. Can be divided into three classes:  Parallel – chains run in the same direction (One side will have either all N or C terminus, R group all point in same directions)  Bottom  Antiparallel – chains that run in opposite direction (one side will have alternating patter of C and N and R groups will either be facing up or down)  Top  Mixed – will contain combination of parallel and antiparallel.  5-10 AA per strand, and can have 2-15 strands. Results in formation of either cylinder or barrel shape or flat planar sheets.  Note that the arrow head points towards the C-terminus  Motifs and domain are part of tertiary of structure. Motif: recurring combination of secondary structure in different region of the protein, ex (helix-turn-helix). It is described in secondary structural terms.  The helix – loop – helix is a Calcium ion binding motif. It is able to hold calcium ion because it has negatively charged Asp, Asn, Thr.  Zinc-finger motif is designed to hold Zn ion. It holds the Zn with help of 2 His pointing towards the Cys which holds the Zn atom. Domain: subunit of 3D structure which describes how a protein will fold into an independent shape. One polypeptide can have more than one domains, it just depends on how many 3D structure it forms. Domains contain secondary structure, thus it could also have motifs as part of it.  Genotype: the genetic information that is encoded into the linear system of the nucleotides within the DNA.  The primary structure of the DNA codes for the primary structure of the protein.  In Eukaryotes genetic info is segregated into different locations like the nucleus, mitochondria and chloroplast. While in prokaryotes it’s present in nucleoid and plasmids which are integrated with cytoplasm.  Gene is a region of DNA which controls a discrete hereditary trait of characteristic (genesis). It is physical and functional unit of hereditary which carries information from one generation to next. It is an entire DNA sequence necessary for the production of a functional protein or RNA Molecule (Molecular view)  DNA codes for a message which is translated into an amino acid sequence of protein.  There is a switch which determines when and when not to transcribe the DNA.  Gene sintony is the order of which genes occur on chromosomes and they are used to control cluster of genes along with individual genes.  Genes are discrete unit, one gene will not overlap with the other gene. The exception to this is the viruses because they have small genome.  The first amino acid in most of the protein was methionine.  Gene NEEDS a start (promoter), stop (terminator) and open reading frame (the part which contains the information for creation of the gene).  Not all the DNA in the organism codes for a gene. There are many other DNA sequences between genes which doesn’t contain information for synthesis of proteins and RNA.  As the species get complicated the gene density decreases. Ex. Humans have gene density of 10% and 90% of the genes is non-coding gene, it was junk.  Aprox 500 gene is the bare minimum for any living organism. Prokaryotic Gene:  It has a promoter – a region to which RNA Pol binds. (non-coding region)  Exon – the region between the start and stop which is transcribed into the RNA and encodes most or all part of the protein. UTRs: 5’ and 3’ UnTranslated Regions, which are transcribed into RNA however, they aren’t translated into the protein. Eukaryotic Gene:  It has exon, promoter, and UTRs. However, it contains Introns which are region of eukaryotic gene that doesn’t code for proteins but is transcribed to RNA and later removed.  Gene expression is the process which involves transcription and translation of a discrete DNA sequence.  There are two types of mRNA formed:  Monocistornic – contains single gene.  Polycistornic – contains more than one gene. (generally in bacteria)  DNA-dependent RNA synthesis is the process by which the RNA is coded from the DNA.  RNA is synthesized from DNA template is complementary to the DNA template  The nucleotides are added from 5’  3’.  RNA synthesis is catalyzed by RNA polymerase. It creates single stranded DNA template and doesn’t require primer.  The products are called primary transcript.  Prokaryotic cells = mRNA (can be translated immediately)  Eukaryotic cells = heterogeneous nuclear RNA (hnRNA) or pre-mRNA. The hnRNA undergoes post-transcriptional process to become mRNA and then it translated into protein.  In transcription the (-) strand (non-coding = template) is used by the RNA Pol to synthesise sense mRNA. However, the (+) strand (coding = non-template) isn’t transcribed since its carries information which the mRNA contains (same polarity, similar base pairs). If (+) strand is transcribed, it results in antisense RNA.  Transcription start site is always labeled +1, and upstream (away from gene) from start site is labeled -1, -2 and so on. While downstream (towards the gene) is +2, +3 and so on.  A DNA-RNA hybrid happens during transcription, however it is short lived. In this the RNA forms complementary base pairs with DNA.  The process of RNA synthesis is self-energizing reaction since the energy required for this process is provided by hydrolysis of the phosphate groups on each nucleotide base. Eukaryotic hnRNA:  In the processing step, the introns are removed through the process of splicing. In this the sugar phosphate backbone is broken into sections where the introns are removed and the exons are ligated together to form ssRNA molecule, which contains the full gene. The processing is done in the nucleus of the cell.  Polycistronic message contains a promoter which controls the transcription of more than one gene. In this ssRNA, there are more than on gene present. The space between each gene is called the ribosome binding site. This site is used to regulate one or all of the protein regulation present on the ss mRNA. The General Transcription in Prokaryotes:  The RNA Pol has the sigma factor attached to it which allows the RNA Pol to attach to the start sequence. Once the RNA Pol has been attached and the transcription has been started, the sigma factor releases itself (after 10-15 nucleotide have been copied) and binds to another RNA Pol. *Note that there are multiple types of sigma factors, each associated with specific promoter region.  In prokaryotes the RNA Pol is a globular protein which consist of 5 subunits with a catalytic site.  Variable regulatory factor: sigma  RNA Pol holoenzyme = core + sigma factor.  The core enzyme consist of:  α-subunit x2 (36,000 Da x 2) – recognize the upstream element (-40 to -70) in DNA.  β-subunit (150,600 Da) – has the polymerase activity (catalyzes the synthesis of RNA) which includes initiation and elongation.  β’-subunit (155,600 Da) – binds to the DNA and maintains the stability of the RNA Pol  ω-subunit (10,000 Da) – polymerase stability and helping the RNA Pol maintain its 3D structure.  Sigma factor (between 7 to 60 different types of sigma factors) is the variable component of RNA Pol holoenzyme. It tells that the specificity of the RNA Pol varies depending on the sigma factor. Sigma 70 (70Da) – general house keeping sigma factor which is responsible for transcribing RNA and respiration. Sigma 32 – heat stress response.  Transcription can be broken down into 3 stages:  Initiation: the binding of RNA Pol to a promoter sequence.  Elongation: addition of ribonucleotide to the RNA  Termination: dissociation of RNA Pol and release of the primary transcript from template.  The control of transcription is important to production of protein within the cell. The is done by controlling the life time of the RNA. Another the way is by increasing or decreasing the transcription and translation.  The template strand is read from the 3’  5’. It’s being read antiparallel and the RNA is being synthesized from 5’  3’.  The variation in the sigma factor allows for the RNA Pol to recognize different set of genes.  Initiation:  The polymerase binds to the double stranded DNA which is closed.  Once the polymerase binds, it melts the duplex DNA resulting in formation of the open complex. In the open complex, the promoter region is exposed.  Then the polymerase catalyze phosphodiester linkages of two rNTPs.  -10 and -35 is the place on the DNA where the sigma 70 would bind. These two are promoter sequences. The one at -10 is TATA sequence known as Probnow box and the one at -35. There is a secondary promoter region known as the UP element which is located -50 nucleotide.  -10 and -35 promoters are the consensus sequences, meaning they are the most common one.  As the diagram above shows, the sigma binds to -10 and -35 region upstream from the start site. At the same time the alpha subunit extends out and binds to promoter sequences that is even further upstream.  Once the RNA Pol binds, during the elongation stage the sigma falls out and the b-subunit is the one that does the catalysis of the mRNA.  Transcription:  Sigma binds to the promoter region.  The DNA double helix opens up and the rudder directs template strand to active site where the addition of rNTP happens to make the complementary RNA is synthesized. Then it gets reattached to the non-template strand.  Initiation is complete once the sigma leaves.  The RNA Pol reads the template strand from 3’  5’ direction, however the RNA is synthesized from 5’  3’ direction.  Once the elongation is finished the resulting product is ssRNA, and the DNA closes back up and the RNA Pol de-attaches from DNA. The termination occurs when a stem-loop (hairpin) structure forms.  The termination process is assisted by the Rho protein (ATPase – helicase) which binds to specific mRNA sequence and moves up mRNA to catch up to RNA Pol, where it causes the formation of stem-loop structure in the mRNA. This structure destabilize the DNA/RNA heteroduplex a
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