Micrb 265 lec 8
What is the central dogma covered in this lecture?
DNA is replicated into DNA, DNA is transcribed into RNA, and RNA is translated into protein.
What kinds of information can DNA encode besides protein-coding genes?
DNA can also encode structural RNAs, non-coding RNAs, and regulatory sequences.
What are the main types of RNA you need to know?
mRNA, tRNA, rRNA, and ncRNA.
What does mRNA do?
mRNA carries the sequence that will be translated into protein.
What does tRNA do?
tRNA brings amino acids to the ribosome and matches its anticodon to the mRNA codon.
What does rRNA do?
rRNA is a structural and functional part of the ribosome.
What is a nucleoside?
A nucleoside is a sugar plus a base.
What is a nucleotide?
A nucleotide is a sugar, a base, and a phosphate.
What is the difference between DNA and RNA sugar?
DNA has deoxyribose, while RNA has ribose with a 2' hydroxyl group.
What bases are found in DNA?
A, T, G, and C.
What bases are found in RNA?
A, U, G, and C.
What is the difference between thymine and uracil?
RNA uses uracil instead of thymine.
Which bases are pyrimidines?
Cytosine, thymine, and uracil.
Which bases are purines?
Adenine and guanine.
How many hydrogen bonds are in A-T base pairing?
Two.
How many hydrogen bonds are in G-C base pairing?
Three.
Why is G-C pairing more stable than A-T pairing?
Because G-C has three hydrogen bonds instead of two.
What does it mean that DNA strands are antiparallel?
The two strands run in opposite directions, one 5' to 3' and the other 3' to 5'.
How should nucleic acid sequences be read?
Always from 5' to 3'.
What does reverse complement mean?
The two strands are opposite in direction and complementary in base sequence.
What does DNA gyrase do?
DNA gyrase introduces negative supercoils into DNA.
What kind of enzyme is DNA gyrase?
A topoisomerase.
Why is supercoiling important?
It helps compact DNA and manage DNA twisting during processes like replication.
What is semiconservative replication?
Each new DNA molecule contains one parental strand and one newly synthesized daughter strand.
What does DNA polymerase do?
It synthesizes a complementary DNA strand.
In what direction does DNA polymerase synthesize DNA?
Always 5' to 3'.
Why can DNA polymerase only add to the 3' end?
Because it needs a free 3' hydroxyl group to add the next nucleotide.
What three things does DNA polymerase need?
A template, a primer, and dNTPs.
Why does DNA polymerase need a primer?
It can elongate an existing strand but cannot start synthesis from nothing.
What are dNTPs?
Deoxynucleotide triphosphates used as building blocks for DNA synthesis.
Where does the energy for DNA synthesis come from?
From hydrolysis of the phosphate bonds in the incoming dNTPs.
What is the origin of replication in bacteria called?
oriC.
How does bacterial chromosome replication usually proceed?
It starts at a single origin and proceeds bidirectionally.
What is a replication fork?
The site where DNA is unwound and new strands are being synthesized.
What is the theta structure?
The intermediate replication shape formed during bidirectional replication of circular DNA.
What does DnaA do?
DnaA binds oriC and causes bending and separation of DNA strands to initiate replication.
What does helicase do in replication?
It separates the DNA strands.
What does DnaB do?
DnaB is the helicase that unwinds DNA at the replication fork.
What do single-strand binding proteins do?
They stabilize separated DNA strands and keep them from reannealing.
What does primase do?
It synthesizes RNA primers.
What is the leading strand?
The strand synthesized continuously toward the replication fork.
What is the lagging strand?
The strand synthesized discontinuously away from the replication fork in short fragments.
What are Okazaki fragments?
Short DNA fragments synthesized on the lagging strand.
Why are Okazaki fragments needed?
Because DNA can only be synthesized 5' to 3', but the two template strands run in opposite directions.
How many primers are needed on the leading strand?
Usually one to start continuous synthesis.
How many primers are needed on the lagging strand?
A new primer is needed for each Okazaki fragment.
Which DNA polymerase mainly synthesizes new DNA in E. coli replication?
DNA polymerase III.
What does DNA polymerase I do?
It removes RNA primers and fills the gaps with DNA.
What does DNA ligase do?
It seals the remaining nicks and joins DNA fragments together.
What enzyme proofreads during replication?
The proofreading subunit of DNA polymerase III, often described as DnaQ.
What does proofreading do?
It removes mismatched newly added bases.
What is the term for removing a mismatched base during proofreading?
Exonuclease activity.
What happens when replication reaches the end region?
Termination proteins stop further replication and topoisomerases help separate the chromosomes.
Why do bacteria not have a telomere problem like eukaryotes?
Because most bacterial chromosomes are circular.
What is transcription?
The synthesis of RNA from a DNA template.
What enzyme carries out transcription?
RNA polymerase.
What is the bacterial RNA polymerase core enzyme?
The catalytic part of RNA polymerase that synthesizes RNA but needs sigma factor to initiate at the right promoter.
What is a sigma factor?
A specificity factor that helps RNA polymerase recognize the correct promoter.
Do sigma factors catalyze RNA synthesis?
No, they do not have catalytic activity.
What is the RNA polymerase holoenzyme?
The core enzyme plus a sigma factor.
Why is the holoenzyme important?
Only the holoenzyme can properly initiate transcription at a promoter.
What is a promoter?
A DNA region where RNA polymerase binds to begin transcription.
Is the promoter transcribed into RNA?
No, the promoter itself is not transcribed.
What is the Pribnow box?
The bacterial -10 promoter element recognized during transcription initiation.
What is the -35 region?
Another promoter element upstream of the Pribnow box that helps RNA polymerase bind.
What strand does RNA polymerase read?
The template strand.
What strand has the same sequence as the mRNA except T is replaced by U?
The coding strand.
How is mRNA related to the template strand?
It is complementary to the template strand.
How is mRNA related to the coding strand?
It has the same sequence as the coding strand except U replaces T.
In what direction is RNA synthesized?
5' to 3'.
Does RNA polymerase need a primer?
No, RNA polymerase can start RNA synthesis without a primer.
What happens after RNA polymerase binds the promoter?
It unwinds the DNA and forms a transcription bubble.
What is a transcription bubble?
The region of locally unwound DNA where RNA is being synthesized.
What temporary hybrid forms during transcription?
An RNA-DNA hybrid.
How does base pairing work in transcription?
G pairs with C, C pairs with G, A pairs with U in RNA, and T in DNA pairs with A in RNA.
What is termination in transcription?
The process where RNA polymerase stops RNA synthesis and dissociates from DNA.
What is a terminator?
A DNA sequence that signals the end of transcription.
Do all terminators work the same way?
No, some require rho factor and some do not.
What is rho factor?
A protein that helps terminate transcription in some cases.
How is bacterial mRNA different from eukaryotic mRNA?
Bacterial mRNA usually has no 5' cap, no long poly-A tail, and little or no processing.
What is a gene?
A linear nucleic acid sequence with a fixed start and endpoint that encodes a polypeptide or functional RNA.
What is the leader sequence?
The region between transcription start and translation start.
What is the trailer sequence?
The region after the stop codon that is still transcribed before transcription terminates.
What is the Shine-Dalgarno sequence?
A ribosome-binding site in the mRNA upstream of the start codon.
Where is the Shine-Dalgarno sequence found?
In the leader region of the mRNA.
About how far upstream is the Shine-Dalgarno sequence from the start codon?
Usually about 6 to 7 bases upstream.
What is the start codon in bacteria?
AUG.
What amino acid is used to initiate translation in bacteria?
N-formylmethionine.
What are the three stop codons?
UAA, UAG, and UGA.
Do stop codons code for amino acids?
No, they signal termination of translation.
What is a codon?
A three-nucleotide sequence in mRNA that specifies an amino acid or stop signal.
What is an anticodon?
A three-nucleotide sequence on tRNA that is complementary to an mRNA codon.
What are sense codons?
The 61 codons that specify amino acids.
Why is the genetic code called degenerate?
Because multiple codons can specify the same amino acid.
What is codon usage bias?
Some synonymous codons are used more often than others, often because matching tRNAs are more available.
What is third-base wobble?
The third nucleotide of a codon is often less strict in pairing, allowing one tRNA to recognize more than one codon.
Why is the reading frame important?
It determines how nucleotides are grouped into codons, which changes the amino acid sequence.
What determines the reading frame in translation?
The start codon sets the reading frame.
How do you identify the correct translation start in an mRNA sequence question?
Find an AUG with an upstream Shine-Dalgarno-like sequence.
What is the first amino acid of most bacterial proteins during translation initiation?
N-formylmethionine.