Chapter 8: Bacterial Genetics and Biotechnology pt 1

Describe the traits of bacterial DNA.

• DNA organized in nucleoid throughout the cytoplasm

• small genome

• circular chromosome (usually), although may have multiple circular and linear chromosomes

• chromosomes replicate and segregate during cell growth

Describe the traits of eukaryotic DNA.

• DNA contained in nucleus, enclosed by nuclear membrane

• wide range of genome size, including very large

• linear chromosomes (in nucleus); mitochondria (derived from bacteria) have one circular chromosome

• chromosomes segregate by mitosis and meiosis after replication during interphase

What are genome, chromosomes and plasmids?

• genome - complete set of genetic info in a cell, double stranded DNA is the genetic material, can be structurally diverse

• chromosomes - essential genes, circular, single copy

• plasmids - extrachromosomal DNA in bacteria, non-essential but useful w/ antibiotic resistance, circular and copy number varies based on plasmid

What are the differences between the eukaryotic genome and the bacterial genomes?

Bacterial genomes

• coding DNA blocked together

• no nucleus

• can replicate and divide/continuous growth and division

Eukaryotic Genomes

• has non-coding btwn coding

• in nucleus

• has interphase replication and short time in mitosis

What is DNA/RNA made out of? What is the structure?

• nucleotides - 5 carbon sugar, phosphate grp and nucleobase

• DNA - encodes genetic information, AGCT

• RNA - transfer of information from gene to protein, AGCU

• antiparallel - chemical directionality (5’ and 3’) two strands have opposing directionality

• complementary - specific base pairing btwn strand - H-bonds, T=A, C=G (triple bond), in RNA its U=A

• helical - double stranded helix, RNA is often single stranded, but can also be double stranded

What is the central dogma?

• the flow of genetic information is in 1 direction

• DNA replication - process of copying the genome

• Genes - functional units in the genome/chromosome used for gene expression (making RNAs and protein)

What happens in DNA replication?

• before cellular division

• duplicated DNA molecules - each daughter cell receives one copy

  • “vertical” gene transfer (parental to daughter)

• plasmids replicated independently of the chromosome

• Steps of DNA Replication: initiation, elongation, termination

DNA Replication: How is DNA made?

• DNA polymerase - uses DNA as a template along w/primer to synthesize DNA

  • can only synthesize 5’ to 3’

  • very accurate w/ proofreading function

• Primase - makes primer for NRA

• Origin of Replication (Ori) - site in genomes where DNA polymerase and other replication proteins bind and begin replication (replicates in both direction)

• Terminus (ter) - site where replication ends

DNA Replication: Initiation

• unwind the helix at origin - creates replication bubble

• add in RNA primer: primase

• load the enzyme for synthesis: DNA polymerase (cannot start by itself needs primase)

DNA Replication: Elongation

• adding base and reading template strand to put in complementary base using DNA polymerase

• adding in dNTP (dATP, dTTP, dGTP or dCTP)

• release pyrophosphate

• form a phosphdiester linkage

• goes in both direction so two new strands of DNA are replicated at each fork

DNA Replication: Termination

• occurs at Ter sites, replication complete

Properties of DNA replication: Bidirectional

• bidirectional - replication forks move in both directions form the origin - fixed sequence in the genome

  • two new strands of DNA are replicated, one at each fork

  • bacterial have circular genomes

  • replication forks meet at a terminus and process is completed

Properties of DNA replication: Semiconservative

• semiconservative - each daughter cell receives DNA that is made up of old (conserved) strand and a newly synthesized DNA

  • DNA polymerase uses both strands of the DNA meaning both strands are copied

  • old strand is a template for new strand and uses complementary base pairing to make a complementary strand that is antiparallel: A=T and G=T

    

Properties of DNA replication: Semi-discontinuous

• semi-discontinuous - a continuous portion (leading strand) and a discontinuous portion (lagging strand)

  • 5’ to 3’

  • replication forks are moving bidirectional

  • leading strands: continuous so forks and replication are moving in the same direction

  • lagging strand: discontinuous short “Okazaki” fragments

  • DNA ligase joins the fragments

What happens in transcription?

• RNA Polymerase: uses DNA as a template to make RNA

  • needs a DNA template strand

  • 5’ to 3’

  • adds RNA bases so its complementary and antiparallel to DNA template

    

• Promoters: sites in genomes where RNAP binds/ starts

• Terminators: sites where RNAP is released/ end

What is the template strand and what is the coding strand?

• template strand: used by the RNA polymerase to build the RNA transcript which is complimentary and antiparallel

• coding strand (non-template): the exact same nucleotide sequence as the resulting RNA transcript, except that thymine (T) in the DNA is replaced by uracil (U) in the RNA and has ribose sugar

Transcription: Initiation requires RNAP holoenzyme - Sigma Factors

• Sigma factors - part of RNA polymerase (RNAP)

  • recognizes promoter and bind the DNA

  • bacteria code for several different Sigma Factors

  • different sigma factors recognize different promoters, different genes are expressed/made into proteins

  • useful for global changes in gene expression such as large physiological changes across the cell

What do eukaryotes use instead of sigma factors?

• transcription factors

• TATA box recruits other transcription factors and brings RNA polymerase

• need it help to find binding spots

How does RNA Polymerase know which strand to use as the template strand?

• DNA strands are complementary, NOT identical

  • different info is encoded in each strand

• either strand can be used as the template

  • location and orientation of the promoter sequence is important

• DNA template strand read 3’ to 5’

Transcription: What is an operon? How is it different in eukaryotes?

• collection of adjacent genes that are all transcribed into a single RNA and under the control of a single promoter

• one promoter for 2 or more genes transcribed

• mostly found in bacteria

• eukaryotes are monocistronic one transcript, one gene transcribed

What is the post transcriptional processing in eukaryotes?

• add 5’ cap

• add poly A tail at 3; end

• splicing - intron regions removed from the mRNA before the next step (e.g. translation)

• one transcript per gene

Transcription: What are the types of RNA products?

• mRNA messenger (yes protein)

• rRNA ribosomal (no protein)

• tRNA transfer (no protein)

• sRNA small regulatory

What happens in translation?

• synthesis of peptides (polypeptides) from a code found on an RNA template - translate nucleotides into amino acids

• ribosomes: enzymes that catalyzes peptide bond formation btwn amino acids

  • large complex of proteins and ribosomal RNAs (rRNA)

  • enzymatic function done by rRNA: ribozyme

  • mRNA serves as the template 5’ to 3’

  • maintains the correct reading frame and aligns amino acids by aligning the tRNA

Translation: What is tRNAs role?

• delivers correct amino acids to the ribosome

• anticodon on tRNA recognizes codon on mRNA

• anticodon is complementary to codon and antiparallel

• code is triplet (3 nucleotides and 1 amino acids)

Translation: What are the 3 steps?

• Initiation - ribosomal binding sites, initiation factors

• elongation - elongation factor

• termination - occurs at a STOP codon

  • no tRNA or amino acids for stop codons

  • termination factors

What is genetic code and how do you read it?

• genetic code - codons in mRNA specify amino acids, triplet code

  • has 64 codons

  • 4 base options at each of 3 codon positions

  • some amino acids have >1 codon: degenerate code

  • 3 codons specify no amino acids: STOP codons

Translation: Reading Frame

• one of 3 ways the code can be read

• Open Reading Frame (ORF, coding region):

  • region on the mRNA btwn START and STOP codons

  • code for the correct peptide is in the ORF, in the correct reading frame

How is translation in prokaryotes and in eukaryotes different?

• Bacterial

  • operons —> polycistronic mRNA

  • Ribosomal bind sites (RBS): special sequence of bases that help tell the ribosome where to align

  • can have multiple RBSs per transcript

  • puts the ribosome in the correct spot to start at an AUG codon

• Eukaryotes — generally have monocistronic mRNA

  • ribosome recognize the 5’ cap structure and find the AUG from there

Compare and contrast gene expression in bact. and euks?

• bacterias do not have nucleus

  • transcription and translation in bacteria are not separate compartments

  • translation begins before mRNA is fully made

• eukaryotes

  • transcription and translation are in separate compartments

What would we need for DNA synthesis in vitro?

• In vitro: the artificial, laboratory-based production of DNA molecules outside of a living organism using chemical or enzymatic methods such as polymerase chain reaction (PCR)

• DNA templates

• DNA primers

• dNTPs

• DNA polymerase

• Buffer + Mg2+

What is polymerase chain reaction (PCR)?

• Denature - 95C for 30 secs to separate strands

• Anneal - 55C for 30 sec primers hybridized

• Synthesis - 72C for 60 sec tag polymerase replicates sequence