Chapter 14 and 15

describe some posttranscriptional modifications to eukaryotic pre-mRNA and their functions

- addition of 5' cap (facilitates binding of ribosome to 5' end of mRNA, increases mRNA stabiltiy, and enhances RNA splicing) - 3' cleavage and addition of poly(A) tail (increases stability of mRNA, facilitates binding of ribosome to mRNA) - RNA splicing (removes noncoding introns from pre-mRNA, facilitates export of mRNA to cytoplasm, allows for multiple proteins to be produced though alternative splicing) - RNA editing (alters nucleotide sequence of mRNA)

describe the 4 different types of introns in eukaryotic RNA

1. Group 1 introns - these are self-splicing (they can catalyze their own removal) 2. group II introns - These are found in genes of bacteria, archaea, and eukaryotic *organelles.* These are also self-splicing, but their mechanism of self-splicing differs from that of group I 3. Nuclear pre-mRNA introns - these include introns located in the protein-encoding genes of the eukaryotic nucleus. The way they are spliced is similar to that of group II, but these are not self-splicing; their removal requires small nuclear RNAs and a number of proteins. (spliceosomal mechanism) 4. Transfer RNA (tRNA) introns - These are found in tRNA genes of bacteria, archaea, and eukarya. their splicing mechanism relies on enzymes to cut and reseal the RNA There are more types but these are the main ones

list the 4 stages of translation

1. tRNA charging 2. initiation, in which the components necessary for translation are assembled at the ribosome 3. elongation of the polypeptide chain 4. termination

How many tRNA's are there?

20 - because the genetic code only encodes 20 AAs?

describe the addition of the 5' cap

As transcription begins, pre-mRNA's '5 end looks like this: 5'-pppNpNpN...3', but rapidly after initiation of transcription, one of the three phosphates on the 5' end is removed and replaced by a guanine nucleotide via a special 5'-5' bond. Then, one methyl group is attached to position 7 of the guanine, and another may be added to the 2' position of the sugars in the second and third nucleotides. An enzyme, among several others, associated with RNA polymerase II is required for this, and since neither of the other RNA polymerases associate with that enzyme, non of the RNA molecules transcribed by them are capped.

The genetic code consists of a sequence of nucleotides in ____

DNA or RNA

T/F the genetic code is completely universal

F - it is almost universal

describe RNA splicing of eukaryotic mRNA

This modification of eukaryotic pre-mRNA takes place in the nucleus, prior to its transport to the cytoplasm. Splicing requires the presence of 3 sequences in the intron. The ends of the intron are called its 5' and 3' spice sites. These sites possess short consensus sequences - most introns in pre-mRNA begin with GU and end with AG indicating their important role in splicing. indeed, changing a nucleotide at either of these sites will prevent splicing. The third sequence important for splicing is the *branch point* which is an adenine nucleotide upstream of the 3' splice site. Mutation of this nucleotide will also prevent splicing. The 5' splice site, branch point, and 3' splice site are collectively called the *splicing code* becaise it determines where splicing will occur. Additional sequences in introns and exons can also affect the process of splicing. Splicing takes place within a spliceosome, which consists of 5 snRNAs, which associate with proteins to form a total of 5 snRNPs (U1, U2, U4, U5, and U6). The intron first cleaves at the 5' splice site and then the free 5' end of the intron folds back on itsself and binds to the branch point, forming a structure called a lariat. This bond is formed via a transesterification reaction. Next, cleavage at the 3' splice site occurs while the 3' of Exon 1 becomes covalently attached to the 5' of Exon 2. The mature mRNA is then exported to the cytoplasm

How does self-splicing work?

This occurs in group I introns, which accomplish this without any help from enzymes or proteins. They do this by forming a secondary strutcure of 9 hairpins. Group II is the other class of self-splicing introns. They also form secondary strucures in addition to a mechanism similar to the spliceosome-mediated mechanism

what evidence disproved Francis Cricks theory that eukaryotic genes are colinear with the proteins that they encode? (meaning every nucleotide had a corresponding amino acid that it encoded)

When DNA was hybridized with mRNA transcribed from it, regions of DNA that did not correspond to the RNA looped out

How do tRNAs attach to amino acids?

all tRNAs have the sequence CCA at the 3' end, and the carboxyl group is attached at the 3' OH group of the adenine (of the CAA). The key to specificity between an AA and tRNA is a set of enzymes called aminoacyl-tRNA synthetases. A cell has 20 aminoacyl-tRNA synthetases - one for each amino acid. Each synthetase recognizes a particular AA as well asl all the tRNAs that accept that AA. Its recognition of an AA is based on charges, size and the R group. The recognition of the tRNAs depends on the nucleotide sequence of the tRNAs.

In _____, a single pre-mRNA can be processed in different ways to produce alternative types of mRNA, resulting in the produciton of different proteins from the same DNA sequence. An example of this is ____

alternative processing pathways alternative splicing

synthesis begins at the ____ end of a new protein, and the protein is elongated by the addition of new AA at the ___

amino carboxyl

Describe the structure of DNA

amino acides have a central carbon, with a hydrogen, an amino group, a carboxyl group and an R group. Amino acids in proteins are joined together by peptide bonds. AA have polarity in physiological conditions - one end has the positive free amino group (NH3⁺) and the other end has a free COO⁻

through wobble, a single ____ can pair with more than one ____

anticodon codon

give an example of how alternative splicing can depend on the type of tissue

calcitonin cleaves at different splice sites depending if its in thyroid cells vs brain cells, resulting in different forms of calcitonin

what is meant by "the genetic code is generally non-overlapping?"

each nucleotide in an mRNA sequence belongs to a single reading frame

many eukaryotic genes contain coding regions called _____ and noncoding regions called ____, or ____

exons intervening sequences introns

ribosomes play a major role in the transfer of _____ from the ____ to the ____

genetic info genotype phenotype

Difference between mRNA transcription in eukaryotes and bacteria

in bacteria, transcription and translation of mRNA takes place simultaneously and side by side in the cytoplasm: while the 3' end of an mRNA is undergoing transcription, ribosomes attach to the shine dalgarno sequence near the 5' end and begin translation. Theres very little opportunity for posttranscriptional modification before translation. In eukaryotes, transcription of pre-mRNA takes place in the nucleus and translation takes place in the cytoplasm, providing an opportunity for the molecule to be modified before translation. Modifications are made to both the 5' and 3' ends as well as to the protein-coding region. It exits the nucleus as mRNA.

What kinds of RNA-RNA interactions are seen in translation?

interactions betewen rRNA and mRNA to keep the mRNA in the ribosome between the codon on the mRNA and the anticodon on the tRNA between the tRNA and rRNA

discuss the presence of introns in eukaryotes vs bacteria vs organelle DNA

introns are common in eukaryote genes, but are rare in bacterial genes. Introns are present in mito and chloroplast genes

describe mRNA roles and structure

mRNA functions as the template for protein synthesis. It carries genetic info from DNA to a ribosome, where it helps to assemble AA in their correct order. In an mRNA molecule, each AA of a protein is specified by a set of 3 nucleotides, called a codon. Both eukaryotic and prokaryotic mRNA contains 3 primary regions: 1. the 5' unsaturated region (5' UTR) or aka the "leader". - this is a sequence of nucleotides at the 5' of the mRNA that does not encode any of the AAs of a protein. In bacteria, this region contains a consensus sequence called the Shine Dalgarno Sequence. 2. protein-coding region - which comprises the condons that specify the AAs of the proteins. This region begins with a start codon and ends with a stop codon 3. 3' untranslated region (3' UTR) aka the "trailer". - this is a sequence of nucleotides at the 3' end of an mRA strand that is not translated into AAs. This sequence affects the stability of mRNA and helps regulate the translation of the mRNA protein-coding sequence

describe the structure of tRNA

many of the nucleotides in a tRNA molecule are complementary, resulting in the characteristic cloverleaf structure (secondary) with 4 major arms: the acceptor arm, the ΤψC arm, the anticodon arm, and the DHU arm. all arms but the acceptor arm consist of a hairpin structure formed by the pairing of complementary nucleotides. In the acceptor arm, instead of a hairpin it forms a stem that includes the 5' and 3' ends of the molecule with CCA and the end of the 3' end where the AA attaches to the tRNA to be brought to the ribosome. The anticodon arm is at the bottom and it has three nucleotides (the anticodon), which pairs with the corresponding codon on mRNA

Beadle and Tatus reasoned that...

mutations affecting a particular biochemical step occured at a single locus that encoded a particular enzyme. This is the one gene, one enzyme hypothesis

Can DNA undergo intron splicing?

no, because they lack the 2' OH group on the sugar

Srb and Horowitz proposed that the biochemical pathway leading to the amino acid arginine has what three steps?

precurser → ornithine → citruline → arginine an auxotrophic mutant cannot synthesize any compount after the step blocked by a mutation

Briefly discuss the processing the role of rRNA

rRNA is processed after transcription in both eukaryotes and prokaryotes. in e. coli It is transcribed into a 30S rRNA precurser, then methylated, cleaved and trimmed to produce the 3 rRNA components of a bacterial ribosome

what is the ribosome size and subunits in eukaryotes?

ribosome size: 80S Subunits: Large (60S) and small (40s)

What is minor splicing?

some introns in the pre-mRNA of multicellular eukaryotes can undergo this type of splicing if introns have different consensus sequences at the 5' splice site and branch point. This process uses a "minor" splicesosome and uses slightly different proteins

How is the processing of mRNA and tRNA different?

tRNA molecules in both bacterial and eukaryotic cells undergo posttranscriptional modification. Most tRNAs are transcribed as a part of larger precursers that are then cleaved, trimmed, and modified to form mature tRNAs. tRNAs must undergo splicing sometimes but their introns are shorter and lack consensus sites and not mediated by a spliceosome like mRNAs are.

What are isoaccepting tRNAs?

tRNAs that have different anticodons but accept the same AA. Wobble allows the anticodon on one type of tRNA to pair with more than one type of codon on mRNA.

tRNA charging is..

the attachment of a tRNA to its appropriate AA. This requires ATP

explain initiation of translation in bacteria

the mRNA first binds to the small 30S subunit of the ribosome, but it can only do so if the the large 50S subunit is not bound to the small subunit - initiation factor-3 (IF-3) binds to the small subunit to prevent the large subunit from binding to it during initiation. IF-I also helps with this by enhancing the disassociation of the large and small subunits. The small subunit recognizes a sequence in mRNA that allows binding (the initiation site), but protein synthesis cannot proceed yet until a ribonuclease is added, degrading all of the mRNA not covered by the ribosome. The intact mRNA strand (containing the start codon) can now be separated from the ribosome and studied. The initiator tRNA, fmet-tRNA attaches to the initiation codon with the help of IF-2, which forms a complex with GTP. All these components form the 30S initiation complex. In the final step of initiation, IF-3 detatches from the small ribosome subunit, allowing the large 50s subunit to join the complex, now called the 70 S initiation complex

Explain the Shine Dalgarno sequence

this is a conssensus sequence in the 5' unsaturated region in prokaryotic mRNA, which serves as the ribosome binding site during translation. It is located 7 nucleotides upstream from the first codon translated into an AA (the start codon). The shine dalgarno sequence is complementary to a sequence found on one of the RNA molecules that make up the ribosome, and it pairs with that sequence during translation. It tells the ribosome where to bind, because it is right in front of the start codon. There is no equivalent consensus sequence in eukaryotes - In eukaryotes, ribosomes bind to a modified 5' end of mRNA.

describe the addition of the poly(A) tail

this process is called polyadenylation. Processing of the 3' end of pre-mRNA requires sequences, collectively termed the polyadenylation signal, located both upstream and downstream of the cleavage site. The consensus sequence AAUAAA (called the poly(A) consensus sequence) is usuallu 11-30 nucleotides upstream of the cleavage site and determines the point at which cleavage will take place. Downstream of the cleavage site is typically a sequence rich in uracil, or U and G. A large number of proteins help to find the cleavage site and cleave the 3' at the cleavage site. Once cleavage is complete, adenosine nucleotides are added without a template to the 3' end. Since the amount of protein not only depends on the amount of RNA transcribed, but also the stability of the DNA molecule, this poly(A) increases the amount of protein synthesized by protecting the 3' end from degredation by exonucleases, and subsequently increasing the time in which the mRNA stays in tact and available for translation