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MICB 201 (35)
Chapter 03-1


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University of British Columbia
MICB 201
Wade Bingle

• Define the terms genome, genotype, phenotype, chromosome and nucleoid in the context of the cell structure and characteristics of prokaryotes. Phenotype – physical features and functional traits of an organism, a reflection of the proteins possessed at any point in time Gene – unit of genetic information, segment of DNA which possesses info specifying the sequence of AA in a protein or the sequence of nt in a stable RNA molecule Genotype – particular set of genes in the organism’s genome Genome – total physical DNA that specifies an organism’s characteristics • Explain the importance of proteins and diversity of protein function to life. Structure, catalysis, regulation, transport, sensing/signalling, force-generation • Describe how genes and proteins are named. Gene: rpsL or rpsL Protein: RpsL • Explain why and how chromosomal DNA is folded and compacted in Bacteria and Archaea. Different packing in Bac and Archaea. In Bacteria, small positively charged polyamines used to neutralize negatively charged phosphates of DNA reducing repulsive forces and helping to compact the DNA. Also, supercoiling (like a telephone cord twisting). Bacteria produce enzymes that can twice DNA into supercoils. The boundaries of each loop of a folded chromosome has anchoring proteins called histone-like proteins. Histones are also positively charged. In Archaea, DNA is also supercoiled and some Archaea use histone proteins (similar to Euk) to compact their DNA. Nucleosome. • Describe the nature of the prokaryotic nucleoid highlighting the differences between Bacteria and Archaea as well as the unique features of the nucleoid in some Planctomyces In Bacteria and Archaea, chromosome refers to the DNA component of the nucleoid. Most prok possess singular circular chromosome, some possess single linear chromosome, some possess segmented genome. Difference not understood why. Chromosome refers to chromosome and plasmids and prophage. • Explain the structural and functional relationships between the linear structure of a prokaryotic protein encoding gene, a mRNA and a protein. • Explain what is meant by gene expression and why genes need to be expressed to have an effect on an organism’s characteristics Genes need to be expressed  mRNA  protein (or tRNA or rRNA). Genotype determines phenotype but to affect the phenotype, the proteins specified by the genes need to be synthesized (from the genes). When the protein is made, the gene is said to be expressed. Gene expression is the process of transcription (and then translation if it was mRNA). Central dogma. Use RNAP and ribosomes. Gene expression depends on many proteins which have the ability to bind to DNA or RNA. Nucleic acid binding proteins tend to be specific. Some only bind to ssDNA, or dsDNA or RNA, etc. • Distinguish between transcription and translation. • Explain the process of bacterial gene expression from the standpoints of initiation, elongation and termination during the processes of transcription and translation. Prokaryotic mRNA is made out of a 5’ UTR containing the ribosome binding site (5 nt), 5 – 9 spacer nt, the initiation/start codon AUG (5’ to 3’), the coding region, the translation termination or stop codon (UGA, UAA or UAG) and 3’ UTR containing the transcription terminator, a region of dsDNA. Region b/t 5’ and 3’ UTR called coding region b/c consists of codons. Nt sequence of mRNA is used by ribosomes as guide to link AA together in a particular sequence resulting in a unique protein. Ribosomes specifically bind to the mRNA at the ribosome binding site to begin translation. Rbs are about 5 bases in size. There are different rbs. After binding at the rbs, with tRNA, ribosomes move along mRNA from 5’ to 3’ joining AA via peptide linkages. Translation can occur at the same time as transcription. Nucleotides are read by the ribosomes and tRNA. The vast majority of prokaryotic proteins begin with AA methionine specified by AUG. Proteins are synthesized from N terminus to C terminus. That means the 5’ end of the mRNA is for the N terminus. Proteins can synthesize methionine at other positions, but translation only starts at the AUG suitably spaced (5-9 nt) from the rbs. Transcription promoter is the dsDNA region where RNAP initially binds to start transcription. It therefore defines the beginning of a gene and is about 50 bp. Bacteria have a single RNAP with 4 core subunits – 2 alpha and 1 beta and 1 beta prime. RNAP exists as a holoenzyme or core enzyme th (different). The core enzyme must associate with a 5 protein called the sigma subunit to form the holoenzyme. Sigma binds to the promoter first, then RNAP binds and slides along to find the promoter sequence. When detected, it stops and transcription is initiated as the sigma falls off. There are many sigma subunits. Control transcription of entire categories of genes by controlling which sigmas are made. In Archaea and Euk, RNAP have multiple subunits and none are capable of recognizing promoters. Instead, transcription factors recognize and bind to sites adjacent to promoters. When the RNAP slides along the DNA, it stops and initiates transcription when it encounters a bound transcription factor. Following promoter is the coding region, largest part of gene that specifies order of AA. 1 kb gene has an average coding region of 900 bp  299 AA. It takes 3 nt pairs (6 nucleotides) in DNA to specify 1 amino acid in a protein. Following RNAP binding to the promoter, RNAP separates the DNA strands to gain access to the template strand and moves 3’ to 5’ synthesizing mRNA 5’ to 3’. Enzyme uses nt sequence of template as a guide for choosing the complementary nt to link together. Termination. Transcription termination is poorly understood in Euk and Arc. Only look at Bac now. Two mechanisms – Rho independent termination and Rho dependent termination. RNA binding protein called Rho which binds to C-rich sequences in the RNA that is not being translated, then slides along RNA in 5’ to 3’ direction, towards the RNAP. When it catches up to the RNAP, it pulls the transcript free of the RNAP and template, releasing it. This happens at sequences that cause RNAP to pause briefly, thus allowing Rho to catch up. Rho-dependent termination can stop transcription when the RNAP has moved beyond the end of the last gene in an operon. Rho-independent termination. RNAP falls off teh template when it is paused over a series of A-U base pairs (which are relatively weak). Pausing is caused by a stem-loop structure in the RNA. The DNA sequences that encode rho-independent transcription terminators can be recognized as palindromes. When the terminator sequence is transcribed into RNA, forms loop – double stranded hairpin like structure called the transcription terminator. Should be after the last codon. • State the general differences between the processes of bacterial and archaeal gene expression. The differences mainly lie in the process of transcription initiation. For bacteria, there is a sigma and the RNAP core enzyme is made out of 2 alpha, 1 beta and 1 beta prime subunit which all join to form one enzyme. It joins with the sigma to form the holoenzyme. The sigma is the one responsible for identifying the promoter region on the dsDNA. The sigma subunit falls off once transcription has started. In the Archaea, the RNAP is different. Archaea and Euk have similar transcription initiation methods (compared to Arc and Bac). In Archaea, the RNAP is made out of several protein subunits and none of them are similar to sigma. There are transcription factors that bind to the sites adjacent to the promoters. When RNAP slides along the DNA, it stops and initiates transcription when it encounters the bound transcription factor. • Explain why transcription and translation of a gene can occur at the same time in prokaryotes but not in eukaryotes and why many copies of the same protein can be simultaneously translated from a single prokaryotic mRNA. Nucleus. • Describe the similarities and differences between prokaryotic protein-encoding genes and those genes that encode stable rRNA molecules. Prokaryotes have a substantial number of genes that encode stable RNA. In Bacteria and Archaea, these genes typically occur in operons. This makes sense because both rRNA and tRNA needed to do protein synthesis, makes sense to have genes for them in the same operon. Initially RNA products are made together as one long precursor RNA. Nuclease enzymes cleave the precursor molecule yielding individual RNA products. • Explain the structure of operons and their significance. In Bacteria and Archaea, common to find genes clustered together in the genome if they specify proteins involved in the same cellular process. These gene clusters are called operons. Genes of operons are transcribed together as a single mRNA initiating at one promoter, but two or more different proteins are translated from the single mRNA. Thus on the single mRNA there wound be two rbs, two AUG and two stop codons. But only one transcription terminator after the 2 stop codon. • Compare and contrast the expression of genes in an operon composed of protein-encoding genes and and one composed of stable RNA genes. • Distinguish between the number of unique protein molecules a prokaryote can synthesize and the total number of protein molecules that may be present in a prokaryotic cell at any point in time. Not all proteins are produced due to gene expression – depends on environment and need. • Using examples explain why different proteins are normally present in a prokaryotic cell in in a different number of copies. Proteins needed in difference amounts. RNAP always needed but DNAP not. • Explain why prokaryotes regulate gene expression. Different proteins needed in different amounts. Don’t want to waste resources. Don’t want to produce something that isn’t required. Waste of energy. If waste energy, growth rate will be slowed. • Distinguish between constitutive and environmentally-regulated genes and gene expression. • Explain two ways the expression of constitutively-expressed genes is regulated in prokaryotes. One way of controlling this is through promoter strength. A strong promoter has a nucleotide sequence that RNAP binds to more strongly than a weak promoter. If it binds more strongly, it is more likely initiate transcription. A weak promoter tends not to be bound as much, that is – most of the time, RNAP will not be found bound there so it won’t be transcribing as much. Isn’t all or nothing event – dynamic equilibrium. Another way is mRNA stability. If mRNA is less stable, it will tend to be degraded at a higher rate. If it’s not around for that long after transcription, the protein may not be able to be made as much. As soon as they are made, mRNAs subject to degradation by ribonucleases (RNases). If more specific protein needed, more of the mRNA must be transcribed. RNase E is important in the first step of mRNA degradation. Different mRNAs have different half lives. Some have only a minute some have an hour. Longer it lasts, more protein can be made. One factor that affects mRNA half life is the nucleotide sequence of the 5’ UTR. This affects RNase E binding presumably, which initiates the process of degradation. rRNA is protected by proteins making up the ribosome. Double stranded RNA is usually not subject to degradation as much. RNase binds to the 5’ UTR and moves 5’ to 3’ making nicks and therefore equally sized mRNA segments. These segments are further processes by other RNase enzymes. • Describe the protein components of the iron acquisition system in E. coli and their function. Iron is important for nearly all life. Usually insoluble as Iron oxides or hydroxides which are more d
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