End of Ch. 7

Ribosomal RNAs

-23S rRNA and 5S rRNA form the core of the large subunit of bacterial ribosome -23srRNA catalyzes formation of peptide bonds between amino acids in prokaryotes

The Reading Frame

-During translation ribosome always "reads" the mRNA in the 5' to 3' direction in sequential sets of 3 ribonucleotides (reading 3 ribonucleotides at a time is known as reading frame) -DNA polymerase and RNA polymerase move along DNA in 3' to 5' direction -Start codon (AUG) establishes the reading frame of the mRNA -leu ser val thr -2nd reading frame has different amino acids, ser ala leu pro -last reading frame gln arg tyr his -in reality only 1 of these three sequences is generated -only mRNA with start codon will be translated into polypeptide

Monocystronic mRNA vs. Polycistronic mRNA

-In prokaryotes, genes are organized into clusters (operons) that are transcribed together into single mRNA because they are under control of the same promoter -prokaryotes have one circular chromosome, dont have enough space for many promoters, many genes share same promoter and are transcribed together into a single long mRNA -majority of genes share same promoter -Operons are found in both prokaryotes and eukaryotes -Operons are also found in eukaryotes, but they are more common in prokaryotes because prokaryotes have one chromosome while eukaryotes have many chromosomes -polycistronic mRNA which is used by ribosome to make 3 proteins -polycistronic mRNA has 3 start codons allowing ribosomes to make 3 different proteins -eukaryotes have more space on their chromosomes for promoters -in prokaryotes translation begins at start codon AUG- methionine -AUG is preceded by ribosome binding site where ribosome binds -in eukaryotes translation also begins at AUG and they also have ribosome binding site where ribosome binds -ribosome binding site in eukaryotes is located at 5' end of mRNA which is signaled by 5' cap

The RNA World Hypothesis

-Proposes that RNA molecules were precursors to current life -2 evidence to support #1 RNA molecules are self replicating #2 ribozymes

RNA is Thought to Predate DNA and Proteins in Evolution

-RNA is Thought to Predate DNA and Proteins in Evolution -As cells evolved, the DNA replaced RNA as more stable molecule for storing genetic information and proteins replaced RNAs as major catalytic and structural components

Ribosome Binding Sites

-Ribosome has binding site for the mRNA and three binding sites for the tRNAs -A P E

Preparing for Translation

-The appropriate amino acid must be coupled to its corresponding tRNA (charging) - performed by aminoacyl-tRNA synthetase -these enzymes catalyze the addition of appropriate amino acid to its corresponding tRNA -adding amino acid to tRNA makes tRNA charged -because there are 20 different amino acids, there are 20 different aminoacyl-tRNA synthetases -here we have tRNA anticodon with 3' ACC 5' -during translation tRNA anticodon binds to mRNA codon 5' UGG 3' -codon encodes tryptophan - tRNA coupled with its amino acid is called charged tRNA -has amino acid attached to its 3' end -uncharged or "empty" tRNA has no amino acid -mRNA codon 5' UGG 3' encodes Trp -tryptophan will be attached to 3' end of tRNA by aminoacyl-tRNA synthetase -addition of amino acid to 3' end of tRNA requires energy of ATP hydrolysis -once amino acid is attached, the tRNA is called charged -charged tRNA has amino acid at 3' end causing tRNA to dissociate from aminoacyl-tRNA synthetase and travels to ribosome

Inhibitory Effects of Antibiotics on Protein and RNA Synthesis

-different antibiotics that inhibit bacteria but not eukaryotic protein or RNA synthesis -by blocking the binding of aminoacyl tRNA to A site of ribosome preventing the transition from initiation to chain elongation preventing peptidyl transferase from making peptide bonds between amino acids blocks translocation stop in translation or block initiation of transcription by binding to and inhibiting RNA polymerase (not exact but she read off slide) dont memorize the antibiotics just know that antibiotics can inhibit bacterial but not eukaryotic protein or rna synthesis

Translation Termination in Eukaryotes

-elongation phase keeps going until ribosome encounters one of three stop codons -stop codon does not encode amino acid it just tells ribosome to stop translating -no tRNA binds to stop codon- UAG -A protein called release factor binds to the stop codon at the A site -release factor tells ribosome to stop translating -Binding of the release factor alters the peptidyl transferase activity, causing it to catalyze the addition of water instead of amino acid to the peptidyl-tRNA -the addition of water to the last amino acid releases polypeptide -ribosome releases tRNA release factor mRNA and dissociates into small and large subunits -these ribosomal subunits can be reused binding to new mRNA making a new protein

The RNA Message is Decoded on Ribosomes

-eukaryotes have ribosomes free floating in cytoplasm and ribosomes attached to RER, membrane bound ribosomes -in contrast to eukaryotes, prokaryotes have only free floating ribosomes -despite that both prokaryotic and eukaryotic ribosomes are made of 2 subunits: large and small -each subunit is composed of rRNA molecules and proteins -in eukaryotes, large subunit is composed of 49 rRNA/ribosomal proteins and 3 mRNA molecules while small subunit is made of 33 rRNA proteins and 1 rRNA molecule -both subunits have more ribosomal proteins than rRNA molecules -prokaryotic ribosomes are similar to eukaryotic ribosomes except they are smaller but each prokaryotic ribosome has two subunit and each subunit is made of ribosomal proteins and rRNA molecules -in eukaryotes, majority of rRNA molecules is produced with the help of RNA polymerase 1 -tRNAs are made by RNA polymerase 3 in eukaryotes -mRNA rRNA and tRNA are all made by RNA polymerase with no roman numeral

Transfer RNAs (tRNAs)

-in addition to mRNA ribosomes and amino acids, we need tRNA -tRNAs are not translated but are needed -Act as adaptors, linking amino acids to codons -tRNA have 2 different sites #1- anticodon -#2 (3' end) amino acid binding site to which amino acid binds -mRNA codon determines what amino acid will be attached to the 3' end of the tRNA -tRNA anticodon 3' AAG 5' binds to the mRNA codon 5' UUC 3' that encodes Phe -according to genetic code, mRNA codon UUC encodes phenylalanine which will be attached to 3' end of tRNA with anticodon AAG -covalent bond forms between amino acid and 3' end of tRNA -hydrogen bonds form between mRNA codon and tRNA anticodon

Protein Modifications

-in order for proteins to be useful to the cell that should undergo modification -all proteins must be folded because only folded proteins are functional whereas unfolded or misfolded are not • Proper folding -some can fold on their own but some need help of proteins called chaperons -many chaperons are heat shocked proteins • Binding of cofactors -many of proteins act as enzymes which require binding of cofactors to them -cofactors help enzymes work better and some cofactors are inorganic metal ions such as iron zinc and copper while some are organic nonprotein molecules called coenzymes which include ????? modified nucleotides like NAD+ FAD NADP+ (photosynthetic organisms) • Glycosylation- adding sugar to protein (glycoproteins and proteoglycans are proteins which have sugar attached to them) • Phosphorylation (kinases- enzymes which add phosphate groups to proteins) -some proteins are activated by phosphorylation while other proteins are inactivated by phosphorylation -GDP binding proteins which become function when entire molecules of GDP binds to them -two categories: monomeric GTPases which are activated by GEF and trimeric GTP binding proteins which are activated by G protein coupled receptor by causing its alpha subunit to exchange guanosine diphosphate or guanosine triphosphate • Association with other protein subunits -if protein is made of two or more polypeptide chains or subunits -the polypeptide chains should come together allowing protein to reach what level of organization -if protein is made of two or more polypeptides the proteins becomes functional after reaching quaternary level

Translation Initiation in Eukaryotes

-initiator tRNA or tRNA charged with methionine binds to small ribosomal subunit with help of proteins called translation initiation factors -small ribosomal unit, initiator tRNA, initiation factors bind to 5 end of mRNA which is found by 5' cap -5' cap is important because it is where small subunit binds to mRNA during translation initiation -small ribosomal subunit moves along mRNA until it encounters start codon AUG

Translation Elongation

-like transcription, translation is divided into 3 stages, initiation elongation (polypeptide chain increases in length) termination #1 tRNA charged with aa 4 enters A site while tRNA at p site has growing polypeptide chain of 3 amino acids #2 this growing polypeptide chain of 3 amino acids uncouples from the tRNA at P site and connects by peptide bond to amino acid 4 which is attached to tRNA at A site -Peptide bond formation between amino acids is catalyzed by peptidyl transferase in eukaryotes and 23S rRNA in prokaryotes; both enzymes are ribozymes, which are found in large ribosomal subunits -ribozymes are enzymes that are ribosomal RNA molecules -most enzymes are proteins but some are ribozymes and one example is peptidyl transferase -tRNA in a site has polypeptide chain of 4 aa but tRNA in p site is uncharged or empty #3 large subunit translocate to the right. The tRNA with growing polypeptide chain moves to P site while uncharged tRNA moves to e site #4 small ribosomal subunit (holding mRNA) to the right in the 5' to 3' direction and this causes uncharged tRNA to be ejected from ribosome -tRNA at p site has polypeptide chain of 4 aa -Back to step one and tRNA charged with amino acid 5 enters ribosome in a site

RNA Molecules are Self-Replicating

-original RNA sequence in blue is template to produce complementary RNA sequence in dark blue -that complementary template then serves as a template to produce that original sequence -rna molecules are able to make copies of themselves , in modern cells they are no longer self replicating

Protein Degradation

-proteins are made and degraded continually -some proteins are degraded by lysosome while short lived and misfolded proteins are degraded by proteasome •Proteasome degrades short-lived and misfolded proteins -proteasomes are cylindrical protein complexes found in nucleus and cytoplasm in eukaryotes made of enzymes called proteases -degrade only those proteins which have ubiquitin attached to them -Ubiquitin ligase attaches ubiquitin to a target protein -ubiquitination or ubiquitylation -ubiquitinated protein is pulled into proteasome where it is degraded into amino acids by proteases. -Amino acids are released and used by ribosomes to make new proteins

Ribozymes

-some rna molecules are ribozymes which are rna molecules that act as enzymes - ribozyme catalyzes the cleavage of another rna molecule -majority of enzymes are proteins while some enzymes are rna molecules called ribozymes

Translation Elongation in Eukaryotes

-translation initiation factors dissociate -large ribosomal subunit binds to small ribosomal subunit making ribosome functional -initiator tRNA charged with methionine is found at p site of ribosome -a site of ribosome is empty and ready to accept tRNA charged with second amino acid -elongation is when polypeptide chain increases in length -Requires proteins, translation elongation factors to deliver charged tRNAs to the A site of the ribosome -mRNA 2nd codon is UUU so tRNA anticodon with AAA binds to mRNA codon forming hydrogen bonds -tRNA is charged with phenylalanine because that is encoded by UUU -tRNA with anticodon AAA and amino acid phenylalanine enters A site of ribosome and forms hydrogen bonds with codon UUU -now both p and a sites are charged with tRNAs -tRNA at p site has methionine and tRNA at a site has phenylalanine -methionine uncouples from tRNA at p site and becomes coupled via peptide bond catalyzed by peptidyl transferase in eukaryotes which is (ribozyme) ribosomal RNA found in the large ribosomal subunit to phenylalanine which is attached to tRNA at a site -tRNA at p site is uncharged whereas tRNA at a site has chain of two amino acids -ribosome translocate along mRNA from left to right (mRNA goes 5' to 3') -uncharged tRNA goes to e site where it is ejected where tRNA with growing polypeptide chain goes to p site -next tRNA with amino acid three enters a site of ribosome

Polyribosomes

-translation is when mRNA is used by ribosome to make protein consisting of amino acid -in order for translation to happen we need mRNA, ribosomes, amino acid but also tRNA amino aceyl-tRNAs-synthetases and various protein factors -polyribosomes are used to translate proteins in BOTH prokaryotes and eukaryotes -polyribosomes bind to same mRNA to produce many identical copies of the same polypeptide from the same mRNA

The Genetic Code: the Connection between mRNA and Amino Acids

-translation: used by ribosomes to make protein -RNA is made of ribonucleotides -protein is made of amino acid -ribosomes read mRNA and know what amino acids to bring together to create a protein -ribosomes always read 3 ribonucleotides at a time • Three ribonucleotides = codon, each specifies one amino acid -20 different a.a • The genetic code tells us which codons encode each of the twenty amino acids -genetic code consists of mRNA codons -there are 64 mRNA codons -there are 4 different ribonucleotides (A U C G) and three ribonucleotides make a codon - 4 x 4 x 4 = 64 codons - among 64 codons, there are three stop codons: UAA, UAG, and UGA -stop codons do not encode an amino acid -stop codon are also known as nonsense, tell ribosome to stop translating -only need one stop codon to make it stop - one start codon: AUG -"tell" ribosome to stop translating and don't encode any amino acids -"tells" ribosome to start translating and encodes amino acid methionine (AUG); know this codon for the exam -protein synthesis always begins with methionine, so the very first amino acid which is added to protein is always methionine -true for prokaryotes and eukaryotes -terminator tells mRNA to stop transcribing -do not confuse terminator and stop codon -terminator is found on template DNA strand and is made of many deoxynucleotides -stop codon is made of 3 ribonucleotides and tells ribosome to stop translating -2 amino acids, methionine and tryptophan are specified by one codon -majority of amino acids are specified by two or more codons for example: proline is specified with 4 different codons that differ in the last base

tRNA Anticodon

tRNA anticodon 1.Consists of 3 ribonucleotides 2.Complementary to the mRNA codon (nitrogenous bases of tRNA codon form h bonds with nitrogenous bases of mRNA codon and mRNA has codon but tRNA has anticodon) (codon does not equal anticodon) 3.tRNA anticodon and mRNA codon are antiparallel -run in opposite directions -while mRNA codon runs in 5' to 3' direction, tRNA anticodon runs in 3' to 5' direction