Information Flashcards
Replication (principles and mechanisms), Transcription (Principles, Regulationa and post-transcriptional modifications), Translation (Principles, Mechanisms)
What is the replisome ?
The entire complex of enzymes that are responsible for DNA replication
Where do replication start in prokaryotes?
OriC or origin of replication
What are okazaki fragments?
The lagging strand cannot be synthesized continously, and must instead be synthesized in fragments, each with its own primer. These are called okazaki fragments
How are DNA synthesized?
With DNA polymerase. The 3’ OH group is the nucleophile and will launch a nucleophillic attack on the fNTP. This creates a bond between the -OH group and a phosphoryl group. DNA polymerase need a primer with an “open” 3’ -OH group to start it’s reaction. They also require a template DNA strand.
What is proofreading?
3’ -> 5’ exonuclease activity. If the wrong base has been added, the polymerase stops and removes the mismatched base pair
What phases of replication are regulated?
Initiation
What are similarities and differences between DNA synthesis and RNA synthesis?
Both are initiated by a nucleophilic attack from the 3’ -OH group to a (d)NTP. DNA polymerase requires a primer while RNA polymerase does not. Both need a template. DNA synthesis begins at OriC, and RNA synthase begins at promotor sequences or TATA boxes. RNA polymerase lack proofreading
Why do RNA polymerase lack proofreading?
Many copies of RNA are generally produced from a single gene, and all RNA will/can be degraded and replaced, so a mistake will be of less consequence than a mistake in DNA replication.
What is a consensus sequence (in relation to transcription in prokaryote)?
The closer the promotor is to the consensus sequence, the better binding and transcription initiation will be. This makes the base expression vary between different genes in the same organism without any inhibitors/promotors proteins
How can transcription be regulated?
Differences in promotor sequence; proteins that facilitate RNA polymerase binding, or inhibit it.
How are transcription terminated?
Either dependant or independent of rho proteins. Rho indepenant termination is done by the formation of a hairpin structure with a terminator (UUUUU) sequence afterwards. The formation of hairpin formation causes the polymerase to fall off. In rho dependent termination, rho protein associates to specific sequences and makes the RNA polymerase release the transcript
What big classes of RNA are there?
mRNA (messenger), tRNA (transport), rRNA (ribosomal)
What RNA polymerases make what kinds of RNAs?
I: pre-rRNA
II: mRNA
III: tRNA and some rRNA
How is transcription initiated in eukaryotes?
TATA-binding proteins bind to the TATA-box, and multiple TFII factors associate to TBP and recruit RNA polymerase. After RNA polymerase is phosphorylated it leaves the TATA box and begins transcription
What are some modifications that eukaryotic mRNA goes through during/post transcription?
Splicing, 5’ methylguanosine cap, polyA tail
How does splicing work?
The spliceosome, which is made up of multiple snRNAs (Snurps). Splicing occurs at conserved GU AG sequences at the 5’ and 3’ end of the intron. The spliceosome helps activate a A residue which will attack the GU sequence and form a lariat structure.
How can one eukaryotic gene give rise to multiple mRNAs?
Alternative splicing
What kinds of reading frames correspond to genes?
Open reading frames with a start codon without a termination codon for at least 50 codons. Longer ORFs often correspond to genes that encode proteins
What is the wobble base?
The third base of the codon and the first base of the anticodon. While the first two are very important for the specificity of the tRNA, the third can sometimes basepair to more than one base; making it possible for one tRNA to recognize multiple codons. This also permits the dissociation of the tRNA from its codon during protein synthesis making it faster. C/A only recognize one codon, G/U two and I can recognize three
What are the steps of protein biosynthesis?
Activation, Initiation, Elongation, Termination, Folding/posttranslational processing
How are amino acids activated?
The ATP facilitated reaction of attaching the amino acid to the tRNA with the correct codon, done by aminoacyl-tDNA synthases
What makes tRNA able to transport amino acid and facilitate translation?
The structure makes it less likely yo be degraded. It has an amino acid arm with a 3’ OH group that an amino acid carboxyl group can form an ester bond with, and it as an anticodon arm that will base pair with codons and thus pair up the correct amino acid with the correct codon.
What do aminoacyl-tRNA synthases do?
Attach amino acids group to the tRNA. Unique aminoacyl-tRNA synthases for each amino acid.
Where do proofreading occur during translation?
When aminoacyl-tRNA synthase attached amino acids to the tRNA. They have active sites where the wrong amino acids will bind and be hydrolysed. Proofreading also occurs between codons and anticodons during the elongation phase.
What is the Shine-Delgarno sequence?
A sequence before the initiation codon in prokaryotes that guide the ribosome to the correct position
How many sites are there in the prokaryotic ribosome?
3; aminoacyl site (A), peptidyl site (P) and exit site (E)
How is the peptide bond between two amino acids formed?
The ester bond between tRNA and the carboxyl terminus activates the carboxyl group, making it possible for it to be attacked by the incoming amino acid and a peptide bond is formed.
What are some posttranslational modification?
Amino and carboxyl-terminal modifications (removal of (f)met), loss of signal sequence, phosphorylation, addition of prosthetic groups (heme), proteolytic cleavage, formation of disulfide bridges
What are houskeeping genes?
Genes that are expressed at more or less constant level. Expression levels often decided by how close to the consensus sequence the promotor is
What types of regulatory proteins are there and what are their functions?
Specificity factors alter specificity of RNA pol for the promotor
Repressors inpede access of RNA polumerase to the promotor
Activators enhance the polymerase-promotor interaction
Where do repressors bind to and where are those sequences located?
Operators, generally near promoter
Describe negative regulation
Bound repressor inhibits transcription
Describe positive regulation
Bound activator facilitate transcription
What is an effector?
A molecular signal or a ligand that binds to the repressor/activator to cause conformational change and either enhance or inhibit its regulatory activity
What are operons?
Genes encoding products that are participants in a set of related processes that are part of a single transcript. This gene cluster + promoter + additional regulatory sequences are called an operon
Describe the regulation of the lac operon
Negative regulation by the Lac repressor (LacI). When allolactose binds to LacI, it causes conformational changes -> binds less -> allows transcription
Positive regulation by CRP+cAMP. When glucose levels are low in cell, [cAMP] is high. cAMP can bind to CRP which will bind near the promotor and enhance transcription. When glucose is present, [cAMP] is lower and will not bind to CRP -> less transcription. Very low transcirption of lac operon without any repressors/activators
How come allolactose can work as an effector for LacI even though LacI represses transcription of enzymes that break down lactose to allolactose?
The repression is not absolute - there is some leakage even though transcription is repressed. So the cell will have a very low level of enzymes that can break down lactose even though it is not present in the cells
Describe the regulation of the trp Operon
Tryptophan both works as an effector for the Trp repressor, so when Trp levels are high, it binds in higher levels to the repressor and changes its conformation so it can bind to DNA, and inhibit transcription. Another level of regulation is attenuation, where if the levels of Trp is high in the cell, an terminator structure will form while the ribosome is translating, causing the transcription to stop
What factors do you need to take into consideration when expressing a eukaryotic gene in prokaryotes?
It cannot contain introns as they do not have splicing. How would translation start as it does not have a Shine-Delgarno sequence?
