week 5- microbial genetics Flashcards
of chromosomes?
Eukaryotic DNA
shape?
Present in?
telomeres present or no?
introns present or no?
Strands?
sugar?
Linear
Present in nucleus
Telomeres
Introns
Multiple chromosomes
Strands twist
deoxyribose
of chromosomes?
prokaryotic DNA
shape?
Present in?
telomeres present or no?
introns present or no?
Strands?
Sugar?
Circular structure
Present in cytoplasm
No telomeres
No introns
Only one chromosome
Bidirectional replication (single origin)
ribose
DNA supercoiling?
- DNA is helical and will form coils
- Supercoiling loosens up the DNA, making it easier to do. separate the two strands
Amino acid structure?
Protein structure?
- central carbon with a carboxyl group (c-terminus), an amino group (N-terminus), and a side chain
- amino acids linked by peptide bonds btwn c and n
Central dogma?
- DNA makes RNA through transcription, and RNA makes polypeptides through translation
what is the replication fork? Replicon? Direction of DNA?
- is where DNA is unwound
- Replicon- portion of the genome that contains an origin and is replicated as a unit
- bidirectional replication from a single origin (circular bacterial. DNA)
helicase
disrupts H-bonds and helps move the replisome
SS DNA binding protein
protects DNA from damage
Topoisomerase
relives twisting of unwound DNA
primase
synthesizes short RNA primers (~10bp) for DNA polymerase
clamp loader complex
holds DNA polymerase at the DNA strand
Tau
binds and organizes R coli replication proteins
leading strand?
Leading - once opened, DNA polymerase adds complementary strands 5 to 3
What does the polymerase require? How many DNA pol does E. col have?
- template, primer and dNTPs
- 5 with pol 3 playing major role in replication
lagging? process?
- lagging strand, prompted by primers and synthesizes short Okazaki fragments
- DnaA protein binds origin, causing bending and separation of strands
- helicase separates strands, SSB binds
- primase synthesizes RNA primer
- lagging and leading made
- DNA pol 1 removes RNA primers and fills in gaps with DNA
- Okazaki fragments are joined by DNA ligase
How does proofreading happen?
- carried out by DNA pol 3, removes mismatch bases from the 3’ end of the growing strand by the exonuclease activity of the enzyme
- not 100% effective
how does termination in E. Coli happen?
- stops when replisome reaches termination site on DNA, catanes form when topoisomerase breaks and rejoins DNA strands to ease supercoiling and recombinase enzyme catalyzes intramolecular crossover that separates the two strands
What is the bacterial RNA polymerase made of?
- core enzyme = 5 polypeptides
- sigma factor - helps core enzyme recognize the start of the genes
- RNA pol haloenzyme= core enzyme + sigma factor (only haloenzyme can begin transcription)
What does RNA pol use in euk and prokaryotes to initiate transcription?
- TATA box
- prib now box
What is the process of initiation of transcription in bacteria? What are the two bacterial promoters?
- sigma factor helps position core enzyme at the promoter
- 35 bps upstream - sigma factor recognizes
- 10bps upstream - where DNA strands start to separate
how does rna pol initate trabscription ?
- the promoter contains a region where the sigma factor recognizes (-35 bps upstream) and a Pribnow box (-10 downstream) where DNA strands start to separate and transcription starts are (+1)
- (-) upstream and before transcription
Where does the repressor protein bind? activator/
- repressor = operator region
- enhancer region
what is the process of transcription elongation?
- after binding, RNA pol unwinds DNA and processes 5 to 3
- forms a transcription bubble - moves with pol as it synthesizes mRNA and w/in bubble a temporary RNA:DNA hybrid formed
- dNTPs incorporated into RNA, complementary to DNA template
Transcription termination process?
Two mechanisms?
- when core RNA pol dissociates from template DNA
- DNA sequences mark the end of the gene and terminator
- intrinsic termination - common - stem-loop formation (
- factor-dependent - rho protein (helicase)
What is stem-loop formation also referred to as?
-rho independent: Transcription is terminated without the involvement of Rho protein
What is the stem-loop process?
- As RNA polymerase transcribes DNA, part of the RNA can fold into a hairpin loop (due to complementary base pairing in the RNA).
- After this loop, there is a stretch of U nucleotides in the RNA, which pairs weakly with a stretch of A nucleotides in the DNA (because U-A bonds are weak).
- This weak bond and the hairpin cause RNA polymerase to pause, making it easier for the polymerase to detach from the DNA, which ends transcription and releases the RNA strand.
How does Rho-dependent termination work?
- Rho protein attaches to a specific spot on the RNA called the rut site.
- Once bound, Rho moves along the RNA toward the RNA polymerase, unwinding the RNA from the DNA.
- When RNA polymerase reaches a stop signal in the DNA, it pauses, allowing the Rho protein to catch up and separate the RNA from the DNA, ending transcription.
This process helps ensure the RNA is released at the right time.
What is an initiation in translation?
- decodes mRNA and linking AA together to form polypeptide
- direction N->C terminal
What is the function of aminoacyl-tRNA synthetases?
- are enzymes that attach amino acids to the correct tRNA (transfer RNA)
- There are at least 20 different synthetases, each specific to one amino acid.
- This ensures that the right amino acid is paired with the correct tRNA, allowing for accurate protein synthesis during translation.
what are the two exceptions in organelles with a reduced genome?
- 3rd postion wobble - eliminates the need for unique tRNA for each codon and decreases effect of some mutations
- Mycoplasm uses stop codon (UGA) to code for glutamine
- Some microbes use 2 rare amino acids: selenocysteine and pyrrolysine
- These are both encoded by codons that typically function as stop codons but are repurposed in microbes through specific mechanisms
What is the bactieral initiator for tRNA? What do archaea and euk use?
- N-formylmethionine-tRNA - adds AUG downstream of Shine Delgado sequences
- AUG start codon on mRNA is the translation initiation codon
- commonly removed post-translational
- methionine-tRNA (MET)p installs at elongation and do not use sd SEUENCE TO LOCATE THE STRAT OF TRANSLATION INSTEAD uses 5’ cap
What is termination in translation?
Termination: when a stop codon is encountered, ribosomes disassemble, and the polypeptide chain is released
- reaches UAA, UAGM and UGA
Central dogma? How is that different in prokaryotes?
- DNA to mRNA (transcription) to protein (Translation)
- In prokaryotes, transcription and translation are coupled and not separated. This allows the cell to make proteins very quickly (polyribosome)
What is protein translocation? two systems used?
movement of proteins from cytoplasm or across the plasma membrane - depends on where made and where folding needed
- sec system and tat system
what is the sec system? and tat system?
- Sec system: generalized secretion pathway - Recognizes signal sequences within the protein to help
translocate a protein from the cytoplasm to the periplasm or external environment - Tat system: specialized secretion pathway for folded proteins
- Recognizes “twin” arginine residues in the protein signal sequence to direct a protein across the cytoplasmic (inner) membrane.
What is secretion? signal peptide?
movement of proteins from the cytoplasm to teh external environment
- N-terminal sequence that directs peptide-specific route -removed after maturation
What is DNA replication?
- The two strands separate, each serving as a template for synthesizing a complementary strand.
how is protein secreted in gram-negative bacteria? two steps vs one step?
- requires crossing two membranes some
- proteins cross in two steps
- First translocation to the periplasm by the sec or tat system
- Second secretion across the outer membrane
- One-step processes involve multiple polypeptides that completely span the periplasm
